Cardiac neuromodulation and methods of using same

ABSTRACT

The present invention relates in general to methodologies for the treatment quenching preconditioning and communication between the intrinsic cardiac nervous system and an electrical stimulus. In particular, the present invention utilizes spinal cord stimulation to alter and/or affect the intrinsic cardiac nervous system and thereby protect the myocytes, stabilize myocardial electrical instability and/or alleviate or diminish cardiac pathologies.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority under 35 U.S.C. § 119(e) of U.S.Provisional Application Serial No. 60/285,176, filed Apr. 20, 2001,entitled “SPINAL CORD STIMULATION APPARATUS AND METHODS OF USING SAME;”U.S. Provisional Application Serial No. 60/291,681, filed May 17, 2001,entitled “SPINAL CORD STIMULATION APPARATUS AND METHODS OF USING SAME;”and U.S. Provisional Application Serial No. 60/295,028, filed May 31,2001, entitled “SPINAL CORD STIMULATION APPARATUS AND METHODS OF USINGSAME,” the contents of which are hereby expressly incorporated in theirentirety by reference.

BACKGROUND OF THE INVENTION Field of the Invention

[0002] The present invention relates in general to methodologies for thetreatment quenching preconditioning and communication between theintrinsic cardiac nervous system and an electrical stimulus. Inparticular, the present invention utilizes spinal cord stimulation toalter and/or affect the intrinsic cardiac nervous system and therebyprotect the myocytes, stabilize myocardial electrical instability and/oralleviate or diminish cardiac pathologies.

BRIEF DESCRIPTION OF THE FIELD OF THE INVENTION

[0003] Recently, the emergence of novel views of the anatomic pathwaysand neural mechanisms involved in the regional control of the heart haveled to the presently claimed and disclosed intrinsic cardiac nervoussystem modalities and treatments. In fact, it has been determined that alevel of processing occurs that permits independent intrinsic cardiac aswell as intrathoracic extracardiac and central spinal integration ofafferent and efferent autonomic influences, and local neuralcoordination without necessarily involving the higher brain centers.This knowledge has led to the development of the presently claimed anddisclosed invention(s). Lathrop and Spooner [24] have postulated that a“hierarchy of control mechanisms among these different elements, andthat they interact as a system of autonomous efferent feedback loopsrather than simply as relay stations subservient to central command.”Indeed, disruption of neuronal circuitry leads to numerous cardiacpathologies. Neuronal interactions that occur within this circuitry orhierarchy modulate different regions of both healthy and diseasedhearts. Thus, the knowledge of this circuitry and methodologies ofmodulating this circuitry (as disclosed and claimed herein) have allowedfor the development and treatment of cardiac pathologies using noveltherapeutic approaches to ameliorate specific cardiac pathologies.

[0004] Regional control of cardiac function is dependent upon thecoordination of activity generated by neurons within intrathoracicautonomic ganglia and the central nervous system. The hierarchy ofnested feedback loops therein provides precise beat-to-beat control ofregional cardiac function. Contrary to classical teaching, studiesundertaken and disclosed in the present specification utilizingelectrophysiological and neuropharmacological techniques applied fromthe level of whole organ to that of neurons recorded in vitro indicatethat intrathoracic autonomic ganglia act in a manner greater than simplerelay stations for autonomic efferent neuronal control of the heart. Ithas been determined that within this hierarchy of intrathoracic gangliaand nerve interconnections, complex processing takes place that involvesspatial and temporal summation of sensory inputs, preganglionic inputsfrom central neurons and intrathoracic ganglionic reflexes activated bylocal cardiopulmonary sensory inputs. The activity of neurons withinintrathoracic autonomic ganglia is likewise modulated by circulatinghormones, chief among them being circulating catecholamines andangiotensin II.

[0005] The progressive development of cardiac disease is associated withmaladaptation of these neurohumoral control mechanisms. Recent dataindicate that conventional therapy of cardiac diseases such asmyocardial ischemia and heart failure exert their beneficial effects notonly on cardiomyocytes directly, but indirectly via the intrinsiccardiac nervous system. The presently disclosed and claimed inventionsof the complex processing that occurs within the intrathoracic nervoussystem, as well as between peripheral and central neurons, will providea basis for understanding the role that the cardiac nervous system playsin regulating not only the normal heart, but the diseased heart.Information derived from research and experimentation of this complexneuronal hierarchy provides for novel therapeutic approaches for theeffective treatment of cardiac dysfunction including protection ofcardiac myocytes and stabilization of myocardial electrical activity bytargeting various populations of neurons regulating regional cardiacbehavior.

[0006] Varying elements within the cardiac neuronal hierarchy exert moreinfluence over regional cardiac function than has been traditionallyunderstood. For example, it is now well recognized that the cardiacnervous system is fundamental to the management of heart failure. Assuch, this nervous system represents a novel and previously unrecognizedtarget for the treatment of heart failure. Control of regional cardiacfunction is dependent upon intrinsic properties of the cardiacelectrical and mechanical tissues as modulated by neural inputs arisingfrom neurons in the intrathoracic autonomic ganglia, spinal cord andbrainstem. Disruptions in neural inputs to the heart or alterations inthe cardiac interstitial milieu can be associated with deleteriouscardiac structural remodeling and, as a consequence, cardiacdysfunction. In the most extreme case, this becomes evident incongestive heart failure. Excessive activation of the intrathoraciccardiac efferent nervous system, as with myocardial ischemia, can evokeventricular dysrhythmias involving changes within the cardiac nervoussystem in addition to alterations in cardiomyocyte ion channel function.Maladaptation of neurohumoral control mechanisms can likewise adverselyremodel the cardiac extracellular matrix.

[0007] The conventional treatment for reducing the frequency andintensity of angina pectoris resulting from myocardial ischemia isanti-ischemic therapy. These therapies are usually based upon restoringthe balance between myocardial oxygen supply and myocardial oxygendemand. Pharmacological agents and revascularization procedures (CABGand PTCA) are conventional treatments for such disease states. Yet thereare a significant number of patients that do not experience adequaterelief of their anginal symptoms with these treatments or are poorcandidates for these therapies. Thus, alternative approaches utilizingdirect electrical activation of neural elements within the spinal cordhave been devised, with the resultant modulation of the intrathoracicneurohumoral milieu thereby eliciting anti-ischemic, antiarryhtymic, andanti-anginal effects.

[0008] A disturbance of the fine balance within the whole cardiacneuraxis can result in dramatic changes in cardiac efferent neuronaloutflow. Experimental studies have been performed to demonstrate thatpathological processes can change the integrative behavior of thecardiac neuraxis. These changes occur when cardiac sensory neurites areactivated intensely and for long periods, as when cardiac tissue becomesdamaged during regional ventricular ischemia. On the other hand, centralprocessing of cardiac sensory output may become deranged leading toconflicting signals that interfere with the maintenance of cardiacfunction. This has led to the proposed scheme that the hierarchy ofcardiac neurons interact effectively if there is an appropriate balancetherein.

[0009] Under normal, physiological conditions stimuli applied to theheart do not elicit marked changes in cardiac efferent neuronal activitybecause central neurons can suppress excessive cardiac sensoryinformation processing. Information has been obtained to support theconclusion that, in the hierarchy of cardiac control, activation ofspinal neuronal circuits modulate the intrathoracic cardiac nervoussystem. Experimental studies have shown that activation of the dorsalcolumns at the T1-T2 segments significantly reduces the activitygenerated by the intrinsic cardiac neurons in their basal conditions aswell as when activated in the presence of focal ventricular ischemiainduced by occluding the left coronary artery. Not only does dorsalcolumn activation modulate the intrinsic cardiac nervous system, but italso modifies the activity of spinal neurons within the T3-T4 segments.In addition, experimental evidence indicates that the central nervoussystem maintains a tonic inhibitory influence over intrathoraciccardiopulmonary-cardiac reflexes. One of the present inventors has alsoshown that reflexes mediated through the middle cervical ganglion areincreased after decentralization. Based on this evidence, it ispostulated that disease processes change the balance between the centraland peripheral neuronal processing of cardiac sensory information. Thus,use of electrical currents to activate spinal neuronal circuits canreverse or halt disease processes of the heart preconditioning theheart—i.e. applying electrical activation prior to disease—also iscontemplated as a means to pro-actively treat a patient with highsusceptibility to cardiac pathologies including arrhythmias.

[0010] Within the hierarchy for cardiac control, neurons of the uppercervical segments modulate information processing in the spinal neuronsof the upper thoracic segments. In human studies, spinal cordstimulation of the C1-C2 spinal segments relieved the pain symptoms inpatients with chronic refractory angina pectoris. Experimental studiesin support of the presently claimed and disclosed invention have shownthat spinal cord activation of the upper cervical segments of the spinalcord suppressed the activity of spinal neurons in T3-T4 segments.Furthermore, chemical stimulation with glutamate of cells in the C1-C2segments also reduced upper thoracic spinal neuronal activity. The uppercervical region is intriguing because it is positioned betweensupraspinal nuclei and spinal circuitry. Neurons in C1-C2 could serve asa filter, an integrator, or as a relay for afferent information, sincethese neurons receive inputs from vagal afferents from the heart.

[0011] Very little information has been published to address underlyingmechanisms explaining how central and peripheral cardiac neurons processcardiac sensory information and interact in the maintenance of adequatecardiac output. The presently claimed and disclosed invention shows thatdisease processes change the balance between the central and peripheralneuronal processing so involved. For instance, when the activitygenerated by cardiac sensory neurons becomes excessive (such as duringfocal ventricular ischemia), cardiac function is profoundly affected,cardiac myocyte protection is reduced and arrhythmias are increased. Adisturbance of the fine balance within the whole cardiac neuraxisresults in dramatic changes in cardiac efferent neuronal outflow. Overthe past 30 years, the anatomy and function of the peripheral cardiacnervous system has been studied, focusing during the last decade on itsintrinsic cardiac component. The classical view of the autonomic nervoussystem presumes that its intrinsic cardiac component acts solely as aparasympathetic efferent neuronal relay station in which medullarypreganglionic neurons synapse with parasympathetic efferentpostganglionic neurons therein. In such a concept, the latter neuronsproject to end effectors on the heart with little or no integrativecapabilities occurring therein. Similarly, intrathoracic extracardiacsympathetic ganglia have been thought to act solely as efferent relaystations for sympathetic efferent projections to the heart. As thepresently claimed and disclosed invention shows, neural control ofregional cardiac function resides in the network of nested feedbackloops made up of the intrinsic cardiac nervous system, extracardiacintrathoracic autonomic ganglia, the spinal cord and brainstem. Withinthis hierarchy, the intrinsic cardiac nervous system functions as adistributive processor at the level of the target organ. Thus, theintrinsic cardiac nervous system plays an important role in thefunctioning of the heart and in its diseased pathologies. This novelinformation thereafter leads to numerous methodologies (some of whichare claimed and disclosed herein for the treatment, preconditioningand/or quenching of disease pathologies through the use of spinal cordstimulation.

[0012] Experimental studies have also shown that pathological processescan change the integrative behavior of the cardiac neuraxis. Thesechanges occur when populations of cardiac sensory neurites are activatedintensely and for long periods of time when local cardiac tissue becomesdamaged during, for instance, regional ventricular ischemia. Thus, undernormal, physiological conditions stimuli applied to the heart do notelicit marked changes in cardiac efferent neuronal activity becausecentral neurons suppress cardiac sensory information processing. On theother hand, central processing of cardiac sensory output may becomederanged during excessive inputs leading to conflicting signals thatinterfere with the maintenance of cardiac function. This has led to thenovel concept that the hierarchy of cardiac neurons interact effectivelyif there is an appropriate balance therein. Fundamental to thishierarchy is its component on the target organ—the intrinsic cardiacnervous system and its influence on the heart.

[0013] Consistent coherence of activity generated by differingpopulations of neurons is indicative of principal and direct synapticinterconnections between them or, conversely, the sharing by suchneurons of common inputs. Such relationships have been identified amongmedullary and spinal cord sympathetic efferent preganglionic neurons, aswell as among different populations of sympathetic efferentpreganglionic neurons. Different populations of neurons, distributedspatially within the intrinsic cardiac nervous system, respond tocardiac perturbations in a coordinate fashion. If neurons in one part ofthis neuronal network respond to inputs from a single region of theheart, such as the mechanosensory neurites associated with a rightventricular ventral papillary muscle, then the potential for imbalancewithin the different populations of neurons regulating various cardiacregions might occur and, thus, its neurons display little coherence ofactivity. In other words, relatively low levels of specific inputs on aspatial scale to the intrinsic cardiac nervous system result in lowcoherence among its various neuronal components. On the other hand,excessive input to this spatially distributed nervous system woulddestabilize it, leading to cardiac arrhythmia formation, etc.

[0014] Thus it is an object of the present invention to use theidentification of the intrinsic cardiac nervous system along with theexperimental data and results to provide methodologies utilizing spinalcord stimulation for the (1) treatment of cardiac disease pathologies;(2) communication between an external point and the intrinsic cardiacnervous system; (3) preconditioning of the intrinsic cardiac nervoussystem in order to promote a protective effect against cardiac diseasepathologies; and (4) quenching aberrant neuronal activity occuringwithin the intrinsic cardiac nervous system.

[0015] This and numerous other objects of the present invention will beappreciated in light of the present specification, drawings, and claims.

SUMMARY OF THE INVENTION

[0016] The presently claimed and disclosed invention encompasses theconcept of an intrinsic cardiac nervous system and the ability tostimulate this intrinsic cardiac nervous through the use of SCS or DCA.The stimulation of this intrinsic cardiac nervous system results in theability to easily and with minimal invasiveness, treat cardiacpathologies either pre-, during, or post-symptom.

[0017] The presently claimed and disclosed invention provides a methodfor protecting cardiac function and reducing the impact of ischemia onthe heart. This methodology includes the steps of: (1) providing astimulator capable of generating a predetermined electrical signal; (2)placing the stimulator adjacent a neural structure capable of carryingthe predetermined electrical signal from the neural structure to theintrinsic cardiac nervous system; and (3) activating the stimulator fora predetermined period of time to generate the predetermined electricalsignal to protect cardiac function and reduce the impact of ischemia onthe heart. In an alternate embodiment of this method, the neuralstructure is a spinal cord.

[0018] The presently claimed and disclosed invention further provides amethod for treating an animal having a cardiac pathology by protectingcardiac function and reducing the impact of ischemia on the heart. Thismethodology includes the steps of: (1) providing a stimulator capable ofgenerating a predetermined electrical signal; (2) placing the stimulatoradjacent a neural structure capable of carrying the predeterminedelectrical signal from the neural structure to at least one of theintrinsic cardiac nervous system and the heart; and (3) activating thestimulator for a predetermined period of time to generate thepredetermined electrical signal to modulate at least one of theintrinsic cardiac nervous system and the heart, and thereby protectingat least one of the intrinsic cardiac nervous system and the heart totreat the cardiac pathology. I an alternate embodiment of thismethodology, the neural structure is a spinal cord.

[0019] The presently claimed and disclosed invention also provides amethod for electrically communicating with at least one of an intrinsiccardiac nervous system and a heart. This methodology includes the stepsof: (1) providing a stimulator capable of generating a predeterminedelectrical signal; (2) placing the stimulator adjacent a neuralstructure capable of carrying the predetermined electrical signal fromthe neural structure to at least one of the intrinsic cardiac nervoussystem and the heart; and (3) activating the stimulator for apredetermined period of time to generate the predetermined electricalsignal to communicate with at least one of the intrinsic cardiac nervoussystem and the heart. In an alternate embodiment of this methodology,the neural structure is a spinal cord.

[0020] Additionally, the presently claimed and disclosed inventionencompasses a method of modulating electrical neuronal and humoralresponses of at least one of an intrinsic cardiac nervous system and aheart. This methodology includes the steps of: (1) providing astimulator capable of generating a predetermined electrical signal; (2)placing the stimulator adjacent a neural structure capable of carryingthe predetermined electrical signal from the neural structure to atleast one of the intrinsic cardiac nervous system and the heart; and (3)activating the stimulator for a predetermined period of time to therebygenerate the predetermined electrical signal to modulate the electricalneuronal and humoral response of at least of the intrinsic cardiacnervous system and the heart. In an alternate embodiment of thismethodology, the neural structure is a spinal cord.

[0021] Furthermore, the presently claimed and disclosed invention alsocalls for a method of activating spinal cord neurons to induce aconformational change in an intrinsic cardiac nervous system. Thismethodology includes the steps of: (1) providing a stimulator capable ofgenerating a predetermined electrical signal; (2) placing the stimulatoradjacent a spinal cord to carry the predetermined electrical signal fromthe spinal cord to an intrinsic cardiac nervous system; and (3)activating the stimulator for a predetermined period of time to therebygenerate the predetermined electrical signal to thereby activate spinalcord neurons in proximity of the stimulator so as to induce aconformational change in the intrinsic cardiac nervous system. In analternate embodiment of this methodology, the neural structure is aspinal cord.

[0022] The presently claimed and disclosed invention also provides for amethod for the prolonged activation of spinal cord neurons to induce aconformational change in an intrinsic cardiac nervous system. Thismethodology includes the steps of: (1) providing a stimulator capable ofgenerating a predetermined electrical signal; (2) placing the stimulatoradjacent a spinal cord to carry the predetermined electrical signal fromthe spinal cord to an intrinsic cardiac nervous system; and (3)activating the stimulator for a predetermined period of time to therebygenerate the predetermined electrical signal to thereby activate spinalcord neurons in proximity of the stimulator so as to induce aconformational change in the intrinsic cardiac nervous system whereinthe activation effects persist for a period of time extending beyond theactivation of the stimulator. In an alternate embodiment of thismethodology, the neural structure is a spinal cord.

[0023] Additionally, the presently claimed and disclosed inventionincludes a method for transiently nullifying neuronal activation of anintrinsic cardiac nervous system by myocardial ischemia. Thismethodology includes the steps of: (1) providing a stimulator capable ofgenerating a predetermined electrical signal; (2) placing the stimulatoradjacent a neural structure capable of carrying the predeterminedelectrical signal from the neural structure to the intrinsic cardiacnervous system; and (3) activating the stimulator for a predeterminedperiod of time to thereby generate the predetermined electrical signalto transiently nullify neuronal activation of an intrinsic cardiacnervous system by myocardial ischemia. In an alternate embodiment ofthis methodology, the neural structure is a spinal cord.

[0024] The presently claimed and disclosed invention also provides for amethod for prolonged nullification of neuronal activation of anintrinsic cardiac nervous system by myocardial ischemia. Thismethodology includes the steps of: (1) providing a stimulator capable ofgenerating a predetermined electrical signal; (2) placing the stimulatoradjacent a neural structure capable of carrying the predeterminedelectrical signal from the neural structure to the intrinsic cardiacnervous system; and (3) activating the stimulator for a predeterminedperiod of time to thereby generate the predetermined electrical signalto nullify the neuronal activation of the intrinsic cardiac nervoussystem by myocardial ischemia for a prolonged period of time extendingbeyond stimulator activation. In an alternate embodiment the neuralstructure is a spinal cord.

[0025] The presently claimed and disclosed invention also includes amethod for transiently suppressing neuronal activation of an intrinsiccardiac nervous system by myocardial ischemia. This methodology includesthe steps of: (1) providing a stimulator capable of generating apredetermined electrical signal; (2) placing the stimulator adjacent aneural structure capable of carrying the predetermined electrical signalfrom the neural structure to the intrinsic cardiac nervous system; and(3) activating the stimulator for a predetermined period of time tothereby generate the predetermined electrical signal to transientlysuppress the neuronal activation of the intrinsic cardiac nervous systemby myocardial ischemia. In an alternate embodiment of the presentmethodology, the neural structure is a spinal cord.

[0026] The presently claimed and disclosed invention includes a methodfor prolonged suppression of neuronal activation of an intrinsic cardiacnervous system by myocardial ischemia. This methodology includes thesteps of: (1) providing a stimulator capable of generating apredetermined electrical signal; (2) placing the stimulator adjacent aneural structure capable of carrying the predetermined electrical signalfrom the neural structure to the intrinsic cardiac nervous system; and(3) activating the stimulator for a predetermined period of time tothereby generate the predetermined electrical signal to suppress theneuronal activation of the intrinsic cardiac nervous system bymyocardial ischemia for a prolonged period of time extending beyondstimulator activation. In an alternate embodiment of this methodology,the neural structure is a spinal cord.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0027]FIG. 1 is a schematic of the neural interactions occurring withinthe intrathoracic autonomic ganglia and between the peripheral networksand the central nervous system. Within the intrinsic cardiac ganglia areincluded sympathetic (Sympath) and parasympathetic (Parasym) efferentneurons, local circuit neurons (LCN) and afferent neurons. Containedwithin the extracardiac intrathoracic ganglia are sympathetic efferentneurons, local circuit neurons and afferent neurons. These intrinsiccardiac and extracardiac networks form separate and distinct nestedfeedback loops that act in concert with CNS feedback loops involving thespinal cord and medulla to regulate cardiac function on a beat to beatbasis. These nerve networks are also influenced by circulating humoralfactors including catecholamines (catechol) and angiotensin II (ANG II).Aff., afferent; DRG, dorsal root ganglia; G_(s), stimulatory guaninenucleotide binding protein; G_(i), inhibitory guanine nucleotide bindingprotein; AC, adenylate cyclase; β_(i)-beta-1 adrenergic receptor;M₂-muscarinic receptor.

[0028]FIG. 2 shows chronotropic (ECG), inotropic (LVP, Left vent.pressure) and neuronal responses recorded simultaneously in atrial(right atrial ganglionated plexus; RAGP) and ventricular (cranial medialganglionated plexus; CMVGP) intrinsic cardiac neurons before and duringtransient occlusion of the left anterior descending coronary artery.Note the enhanced activity in both ganglionated plexi, with theventricular ganglionated plexus being more affected.

[0029]FIG. 3 is a graphical representation of the change in intrinsiccardiac neuronal activity induced by transient occlusion of the leftanterior descending artery (CAO) and/or dorsal cord activation (DCA) at90% Motor Threshold.

[0030]FIG. 4 is a graphical representation of long-term effects (memory)on intrinsic cardiac neuronal activity induced by short-term DCA.Following bilateral transection of the ansae subclavia, DCA no longeraffected activity within the intrinsic cardiac nervous system.

[0031]FIG. 5 shows activity generated by two different populations ofintrinsic cardiac neurons contained within the right atrial ganglionatedplexus. Arrow indicates application of veratridine to the epicardium ofthe left ventricle. At baseline, note the cycling of activity with aperiodicity of 20 seconds. In the unstressed condition, this bursting isusually associated with increased coordination of activity between thetwo populations of neurons (see bottom trace). When an afferent stressis imposed to the ICN, as with application of epicardial veratridine,activity increased in both sites and the coherence of activity generatedby these two populations of neurons approached unity.

[0032]FIG. 6 shows that chronic myocardial ischemia is induced byplacement of an ameroid constrictor on the left circumflex (LCx) artery4 weeks previously (panel A). Under basal conditions, electrogramsdisplay slight ST segment displacement (panel B). Transient rapidventricular pacing (240/min for 1 min), used to increase myocardial O₂demand, precipitates ischemic episodes. In the first beats followingrapid pacing, ST segment displacement is inhomogeneously augmented inthe LCx territory. Marked ST segment depression (−2 to −6 mV) occurs insome areas, whereas ST elevation (+2 to +15 mV) develops in others(panel C). ST segment changes were also induced by ANG II whenadministered to RAGP neurons via the right coronary artery proximal tobranching of the SA node artery (40 μg/min for 2 min). Note that the STchanges, induced by ANG II, occurred at the apical margin of the plaqueelectrode, i.e. at the periphery of the LCx territory (Panel D). Incontrast, the changes induced by transient rapid pacing occurred at amore central location in the LCx territory (panel C).

[0033]FIG. 7 shows ST segment changes were induced by angiotensin II(ANG II) administered to RAGP neurons via the right coronary arteryproximal to branching of the SA node artery (40 μg/min for 2 min). Notethat the ST changes occurred at the apical margin of the plaqueelectrode (panel B). Thus, the ST segment changes are caused by director indirect activation of ganglionated plexus neurons that projectefferent axons to the specific ventricular areas in which the changesoccurred. Moreover, the ANG II effects are attenuated by DCA (panel C),showing that such ventricular events can be influenced by interactionsbetween intrinsic cardiac and spinal neurons.

[0034] FIGS. 8A-8C show ISF, aorta and coronary sinus norepinephrine(NE) and epinephrine (EPI) levels in response to stellate stimulation (4Hz), angiotensin II (ANG II) infusion (100 μM, 1 ml/min) into the bloodsupply for the Right Atrial Ganglionated Plexus (RAGP) and Dorsal CordActivation (DCA, 50 Hz, 200 μsec, 90% motor threshold). ISF fluids weresampled using the microdialysis techniques summarized in Aim 3.

[0035]FIG. 9 shows the effects of acetylcholine (ACh) on canineintrinsic cardiac neurons obtained from sham control (CONTROL) and fromhearts where all extracardiac nerve connections to the heart wereinterrupted 3 weeks previously (DCX). The horizontal bar under thetraces indicates application of a 10 ms pulse of ACh (1 mM) from the tipof a pipette placed near the ganglion. A, CONTROL. ACh depolarized acontrol intrinsic cardiac neuron, evoking a short burst of APs at thestart of depolarization. DCX. ACh depolarized the chronicallydecentralized neuron more than the control one, evoking a longer lastingburst of APs. During the repolarization phase, the membrane potentialbegan to oscillate with APs being discharged on oscillatory peaks. B.Hexamethonium (100 μM for 5 min in perfusate) reduced the amplitude ofACh-induced depolarization relative to the control state; no APs weregenerated. DCX. Hexamethonium reduced the amplitude of ACh-induceddepolarization; AP discharge was facilitated during plateau phase ofresponse. The bursting of activity is reflective of the enhancedmuscarinic receptor-mediated responses of these neurons.

[0036]FIG. 10 shows inhibition of ACh-evoked responses by substance P(SP). Top trace shows intracellular recording from an intracardiacneuron and bottom marks indicate times when 28 ms puffs of ACh (10 mM)were given by local pressure injection. Local application of ACh evokedaction potentials. These ACh evoked potentials were blocked during bathapplication of 10 μM substance P (see horizontal bar).

[0037]FIGS. 11A and 11B show photomicrographs showingCGRP-immunoreactive nerve fibers in a dog intracardiac ganglion (panelA) and PGP 9.5-immunoreactive nerve fibers in dog sinoatrial node (panelB). The chromogen was VIP in A and diaminobenzadine in B (both fromVector). Both panels are at the same magnification. Scale bar=50 μm.

[0038]FIGS. 12A and 12B show enhancement of the activity generated by acanine nodose ganglion afferent neuron following application of the longacting adenosine agonist CPA (via a 1 cm×1 cm pledget) to the ventralleft ventricular epicardium (between panels A & B). Monitored cardiacvariables were not affected by this intervention. Panel B was obtained 1minute after terminating CPA application.

[0039]FIGS. 13A and 13B show simultaneous recordings of activitygenerated by intrinsic cardiac (above) and intrathoracic extracardiac(left middle cervical ganglion-LMCG) neurons concomitant with leftventricular sensory inputs induced by epiacrdial application ofveratridine. The right hand panels denote XY plots of each activityversus pressure. Note that enhancement of their ventricular sensoryinputs depicted in panel B activated one population while suppressingthe other. Activities ocurred during specific phases of the cardiaccycle (XY plots).

[0040]FIG. 14 shows examples of two different pairs of spinal neurons inthe T3 spinal segment. Aa is background activity recorded from deeper(Unit 1; lamina V-VII) and superficial (Unit 2; lamina I-II) neurons. Abis the cross-correlogram of the background activity. Central peakscentered around 0 delay represent the action potentials that occur fromone neuron shortly before (negative delays) or after an action potentialoccurs in the other neuron. Ba is activity from superficial (Unit 1;lamina I-II) and deeper (Unit 2; lamina V-VII) neurons evoked by aninjection of bradykinin into the pericardial sac. Bb is thecross-correlogram of the evoked activity. The upper tracings aredischarge rate in impulses/sec (imp/s) and lower tracings (Unit) are theraw records of the extracellular action potentials. The arrows representthe injection (upward) and removal of bradykinin. The characteristics ofthe cross-correlograms were similar to those described by Sandkuhler etal.

[0041]FIG. 15 shows responses of a T3 spinal neuron to visceral andsomatic stimulation. A&B: responses of the cell to saline (A) and tointrapericardial injections of algogenic chemicals before (A) and after(B) the spinal cord was transected at the C7 segment. C: responses ofbrushing hair (Br) and pinching (Pi) the skin in the somatic fieldrepresented by the ellipse on the rat figurine. D: the black dot marksthe location of the recording site for this cell.

[0042]FIG. 16 shows response of T3 deeper spinal neuron to occlusion ofthe left coronary artery (CAO). The top trace is the rate of celldischarges in impulses/ sec (imp/s). The second trace shows the rawtracing of the individual extracellular action potentials (CellActivity). The third trace is blood pressure in mmHg. The horizontal barrepresents the stimulus period for CAO. The occlusion was sustained forone minute.

[0043]FIG. 17 shows intrapericardial infusion of algogenic chemicalscaused intense c-fos immunoreactivity in the nuclei of T3-T4 neurons(arrows) in the marginal zone (left photo) and central gray region(right photo; cc—central canal).

[0044]FIG. 18 shows distribution of c-fos immunoreactive (IR)neurons/100 μm in the C1 spinal segment following (A) unoperatedcontrol, (B)—Vagal crush, (C) Vagal stimulation. Following stimulationof the vagus, c-fos IR neurons (black dots) were abundant in the medialmarginal zone and substantia gelatinosa. C-fos IR neurons also werelocated throughout the nucleus proprius, along the marginal zone, in theventral horn, and central gray region.

[0045] FIGS. 19A-19F show responses of T3 cell to chemical stimulationof glutamate before and after rostral C1 spinal transection. Theresponses were evoked by intrapericardial injections of bradykinin (BK).Saline was used as the control. Pledgets of glutamate placed on theC1-C2 dorsal spinal cord (B) decreased the discharge rate of the cellfor the three minute period it was applied. The background activityrecovered after glutamate was removed. After the rostral C1 cut, BKstill increased the discharge rate of the thoracic STT cell (D) althoughthe BK response characteristics changed. The presence of glutamateattenuated this response (E). The increased rate of discharge to BKinjections was again observed when glutamate was removed (F). In eachpanel, action potentials were recorded on a rate histogram.

[0046]FIG. 20 shows anterogradely labeled fibers (arrows) with PHAL wereabundant in the T3-T4 central gray region (area X; photograph on right;cc=central canal). The lateral portion of the central gray regioncontains the intermediomedial (IMM) cell nucleus where somepreganglionic sympathetic neuronal cell bodies reside. Abundant PHALimmunoreactive fibers also were found in the superficial dorsal horn andnucleus proprius (photograph on left) in the T3-T4 segments.

[0047] FIGS. 21A-21C shows effects of vagal afferent stimulation on thebackground activity and evoked activity of a T3 neuron before ibotenicacid was placed on the dorsal C1-C2 spinal cord. Electrical stimulationof the left cervical vagus (A: LCVS; {30 V, 0.1 ms} ipsilateral to thecell) right cervical vagus (B; RCVS; contralateral) at differentfrequencies. Vagal stimulation reduced the discharge rate of the evokedresponse to noxious stimulation of the cardiac afferentsintrapericardial injections of bradykinin (C). IA; ibotenic acid. Theshort horizontal bars represent the period of vagal stimulation, thelong horizontal bar represents the bradykinin injection and the numbersindicate the frequencies tested.

[0048]FIG. 22 shows vehicle (A) or ibotenic acid (B) was placed viapledget on the dorsal surface of the C1-C2 spinal segments for 2 hrs.After 14-16 hrs, rats were perfused with fixative and the medulla, C1-2,C3-5 segments were processed for annexin fluorescence histochemistry.Photomicrographs are from the C1 dorsal horn and the gray matter isoutlined. Very little annexin staining was observed in control tissuesections (A) or in the medulla and C3-5 segments from ibotenic acidtreated rats. White arrows point to unlabeled (black) cells in thedorsal horn. In rats treated with ibotenic acid (B), many annexinpositive (white) cells were observed (arrows). Annexin binding indicatescells with energy impairment and/or undergoing apotosis. Annexin belongsto a family of proteins that bind acidic phospholipids, particularlyphosphotidylserine (PS). PS is assymetically distributed in the cellmembrane by the enzyme, aminophospholipid translocase. Following energyimpairment, PS distributes to the outer cell leaflet and annexin bindingillustrates cells with PS on the outside of the cell.

[0049] FIGS. 23A-23C show responses of T3 cell to intrapericardialinjections of bradykinin (BK) before and after dorsal cord activation.Electrical stimulation (250 μA, 0.25 us and 50 Hz) of the ipsilateral(A) or contralateral (B) C1-C2 dorsal columns applied prior tointrapericardial injections of BK markedly reduced the evoked responses.C: dorsal cord activation during the evoked response to BK also reducedthe cell activity. Horizontal lines are the period of the stimulus.

[0050]FIGS. 24A and 24B show epicardial conduction mapping across theanterior myocardial infarction in a susceptible dog (panel a) and aresistant dog (panel b) with normal left ventricular function. Thelongest time for epicardial electrical activation was about 80milliseconds in susceptible dogs. This is in contrast to resistant dogsin which the longest time for epicardial activation was about 40milliseconds.

[0051]FIG. 25 shows stratification of ventricular fibrillation risk in asusceptible dog. Left panel illustrates induction of ventricularfibrillation during exercise and myocardial ischemia test. As shown inright panel, susceptible dogs are characterized by a tachycardicresponse to acute myocardial ischemia that is uncontrolled and leads toVF. Resistant dogs have an increase in heart rate within 15 seconds ofcoronary occlusion, but have strong vagal reflexes that reduce heartrate within 30 seconds of the occlusion as illustrated in the rightpanel.

[0052]FIG. 26 shows the heart rate slowing in response to systemichypertension (phenylephrine induced) quantifies baroreflex sensitivity.

[0053]FIG. 27 shows chronotropic response to graded increases intreadmill exercise. Once heart rate reaches 210 beats per minute thecircumflex occluder is inflated for 2 minutes, the first minute the dogscontinue to run on the treadmill and the treadmill is stopped for thelast minute. While concurrent DCA minimally affected heart rateresponses in the resistant dog (right panel), in the susceptible dog DCAreduced the heart rate during the ischemic period (left panel).

[0054]FIG. 28 shows heart rate variability was computed from 25 minutesof continuous resting ECG with spectral densities computed using a fastFourier transformation. High frequency variation in heart rate isthought to predominantly arise from vagal input to the SA node, whilelower frequency bands (VLF, LF) are thought to arise predominantly fromsympathetic activity. DCA effects were examined on two different daysfollowing 4 days of stimulation lasting for 4 hours.

[0055]FIG. 29 shows dorsal cord activation increased the standarddeviation of the RR intervals in both resistant and susceptible dogs,again suggesting that cardiac autonomic neuronal activity shifted towardefferent vagal control. Standard deviation of the RR interval valuesbelow 100 milliseconds predicts high risk for ventricular fibrillationduring exercise and ischemia.

[0056]FIG. 30 shows percent change is ISF NE and EPI in response to solecoronary artery occlusion (CAO, solid lines) and CAO in the presence ofDCA (CAO+DCA, dotted lines). Left panels show data from normallyperfused left ventricular regions. Right panels show data from theischemic zone. Time 0 is pre-occlusion baseline; CAO is on for 15 min.

[0057]FIG. 31 shows effects of DCA on induction of ventricularfibrillation (VF) associated with 15 min coronary artery occlusion andreperfusion. Arrows indicate time point for onset of VF. With coronaryartery occlusion, VF was induced in 50% of the animals; when VFoccurred, it was within 6 min of reperfusion onset. With pre-existingDCA, coronary artery occlusion induced VF in only 1 of 9 animals (1 minpost DCA; 7 min post-occlusion).

[0058]FIG. 32 shows examples of the activity generated by a pair ofsuperficial spinal neurons in the T3 spinal segment. Aa is basalactivity recorded simultaneously from the neuron pair with intactneuraxis and Ba is basal activity after vagotomy. With the neuraxisintact, the cross-correlogram of the basal activity between these twoneurons showed a central peak centered around 0 delay and a secondsmaller peak occurred approximately 150 ms after the central peak.Following bilateral vagotomy, the central peak was reduced and thesecondary peak eliminated. Upper tracings represent discharge rate(impulses/sec; imp/s) and lower tracings (Unit) extracellular actionpotentials.

[0059]FIG. 33 shows responses of a T3 spinal neuron to an electricallyinduced premature ventricular contraction. The extra stimulus wasdelivered at the arrow in the top trace. This stimulus produced apremature ventricular contraction that was followed by a compensatorycontraction (CC in middle trace). The 2^(nd) arrow in the top tracepoints out the burst of neuronal activity following the extra stimulusthat was associated with the potentiated beat. The arrow in the bottomtrace indicates electrical activity associated with the electricalstimulus. The ECG was recorded from lead II.

[0060]FIG. 34 shows the average neuronal activity data derived from allanimals during each of the five protocols utilized in this study. WhenSCS was applied alone (A) neuronal activity was suppressed, a changewhich persisted for a short time after terminating the SCS (SCS off).(B) Coronary artery occlusion (CAO) enhanced neuronal activity. (C) SCSsuppressed neuronal activity before, during and after coronary arteryocclusion. Data obtained for the other protocols (SCS and CAO) arepresented in panels D and E. * Represents data which was significantlydifferent from control values (P<0.05).

[0061]FIG. 35 shows the initiation of coronary artery occlusion (arrowbelow) resulting in an increase in the activity generated by rightatrial neurons (individual units identified by action potentials greaterthan the small atrial electrogram artifacts). From above down are theECG, aortic pressure (AP), left ventricular chamber pressure (LVP) andneuronal activity. Horizontal timing bar=30 s.

[0062]FIG. 36 shows the influence of SCS on the ECG, left ventricularchamber pressure (LVP=145 mmHg) and intrinsic cardiac neuronal activity(lowest line) before and during coronary artery occlusion. (A) Multipleneurons generated action potentials, represented by their differingheights, at a rate of 132 impulses per minute (ipm) during controlstates. (B) Once SCS was initiated (note stimulus artifacts in theneuronal tracing), neuronal activity decreased to 34 imps/min (noactivity generated during the record). ECG alterations were inducedthereby. (C) Neuronal activity continued at the rate (39 imp) in thepresence of SCS even though coronary artery occlusion had beenmaintained for over 1.5 min.

[0063]FIG. 37 shows the transmural blood flow (ml/min/g) to LV ischemic(closed spheres) and non-ischemic (closed squares) zones for each of thethree baseline control conditions (C1, C2, and C3) and during thesuccessive interventions of 5-min spinal cord stimulation (SCS), 4-minocclusion of the LAD occlusion commencing 1 min into SCS (SCS-CO).Transmural blood flow within the ischemic zone is significantly lower (*p=0.02) during both CO, and SCS-CO (p=NS between these twointerventions) compared to base line.

[0064]FIG. 38 shows presure-volume (P-V) loops for the left ventricle;P-V loops obtained under basal conditions are shown in panels (A), (C)and (E) (i.e., baseline steady-state resting conditions). P-V loopsobtained during SCS at 90% motor threshold (B), 4 min of LAD occlusion(D), and concurrent SCS and LAD occlusion (F) are also shown.

[0065]FIG. 39 shows a graphical representation of the two protocols ineach group of five dogs. Note that 1.5 h was allowed to lapse betweeneach intervention in either protocol.

[0066]FIG. 40 shows the effects of coronary artery occlusion on theactivity generate by intrinsic cardiac neurons in one animal. Followingocclusion of the left anterior descending coronary artery (beginning atarrow below), the activity generated by right atrial neurons (lowestline) increased (right-hand panel). Heart rate was unaffected by thisintervention, while left ventricular chamber systolic pressure (LVP)increased a little. The time between panels represents 1.5 min.

[0067]FIG. 41 shows the activity generated by intrinsic cardiac neuronsin one animal during control states (panel A, lowest line) decreasedwhen the dorsal aspect of the spinal cord was stimulated (panel B). Thesuppressor effects of SCS persisted during coronary artery occlusion(panel C). The electrical stimuli delivered during SCS are representedin panels B and C by regular, low signal-to-noise artifacts (note thatatrial electrical artifact is recorded during each cardiac cycle as alow signal during the p wave of the ECG). The suppression of spontaneousactivity generated by intrinsic cardiac neurons persisted afterdiscontinuing SCS (panel E represents neuronal activity recorded 5 minpost-SCS and 6 min post-LAD occlusion; panel D represents basal activityat same time scale obtained before commencing these interventions).ECG=electrocardiogram; AP=aortic pressure; LVP=left ventricular chamberpressure.

[0068]FIG. 42 shows representative ECG records obtained from one animalduring control states (A), as well as a few minutes after beginningcoronary artery occlusion in the presence of spinal cord stimulation (B)and at the end of occlusion while SCS was maintained (C). Note that STsegment alterations occurred throughout the period of ischaemia.

[0069]FIG. 43 shows the average neuronal activity recorded in allanimals before, during and after dorsal spinal cord stimulation (SCS)delivered in the presence of coronary artery occlusion (occlusion). Notethat SCS reduced neuronal activity soon after its application began. SCSalso prevented enhancement in intrinsic cardiac neuronal activitynormally associated with coronary artery occlusion (cf. Table V).Neuronal activity remained reduced for 17 min after terminating SCSdespite the induction of myocardial ischaemia. These data were collectedduring application of the first SCS in protocol 2.

DETAILED DESCRIPTION OF THE INVENTION

[0070] Before explaining at least one embodiment of the invention indetail, it is to be understood that the invention is not limited in itsapplication to the details of construction and the arrangements of thecomponents set forth in the following description of illustrated in thedrawings. The invention is capable of other embodiments or of beingpracticed or carried out in various ways. Also, it is to be understoodthat the phraseology and terminology employed herein is for purpose ofdescription and should not be regarded as limiting.

[0071] The intrinsic cardiac nervous system has been classicallyconsidered to contain only parasympathetic efferent postganglionicneurons that receive inputs from medullary parasympathetic efferentpreganglionic neurons. As such, intrinsic cardiac ganglia have beenviewed as simple relay stations and major autonomic neuronal control ofthe heart was believed to reside solely in the brainstem and spinalcord. However, the data supporting the presently claimed and disclosedinvention indicate that centripetal as well as centrifugal processingoccurs within the mammalian intrathoracic nervous system (i.e. theintrinsic cardiac nervous system). This involves afferent neurons, localcircuit neurons (i.e., neurons that interconnect neurons within oneganglion and neurons in different intrathoracic ganglia), as well assympathetic and parasympathetic efferent postganglionic neurons.

[0072] The intrinsic cardiac nervous system consists of multipleaggregates of neurons and associated neural interconnections, localizedto discrete atrial and ventricular regions. Among these distinctganglionated plexi, preferential control of specific cardiac functionshas been identified. For example, right atrial ganglionated plexusneurons have been associated with primary, but not exclusive, control ofSA nodal function and inferior vena cava-inferior atrial ganglionatedplexus neurons primarily, but not exclusively, with control of AV nodalfunction. One population of intrinsic cardiac neurons, theparasympathetic postganglionic ones, receives direct input frommedullary parasympathetic preganglionic neurons. Another population,adrenergic efferent ones [8,9], receives input from more centrallylocated neurons in intrathoracic ganglia and the spinal cord. The factthat ventricular sensory neurites continue to influence the activitygenerated by neurons on the heart following chronic decentralization ofthe intrinsic cardiac nervous system indicates that the somata ofafferent neurons, some of which project axons to central neurons, arelocated within the intrinsic cardiac nervous system. This concept hasreceived anatomical confirmation. Functional data also indicate that theintrinsic cardiac nervous system contains local circuit neuronsinterconnecting intrinsic cardiac afferent with efferent neurons.

[0073] Sub-populations of right atrial neurons that receive afferentinputs from sensory neurites in both ventricles are responsive to localmechanical stimuli and the nitric oxide donor nitroprusside. Neurons inat least one ganglionated plexus locus were activated by epicardialapplication of veratridine, bradykinin, the β1-adrenoceptor agonistprenaterol or the excitatory amino acid glutamate. Epicardialapplication of angiotensin II, the selective β₂-adrenoceptor agonistterbutaline or selective α₁- or α₂-adrenoceptor agonists elicitedinconsistent neuronal responses. The activity generated by bothpopulations of atrial neurons studied over 5 minute periods during basalstates displayed periodic coupled behavior (cross correlationcoefficients of activities that reached, on average, 0.88±0.03; range0.71-1) for 15-30 seconds periods of time. These periods of coupledactivity occurred every 30-50 second during basal states, as well aswhen neuronal activity was enhanced by chemical activation of theirventricular sensory inputs. It has been observed that neurons throughoutone intrinsic cardiac ganglionated plexus receive inputs from mechano-and chemo-sensory neurites located in both ventricles. That such neuronsrespond to multiple chemical stimuli, including those liberated fromadjacent adrenergic efferent nerve terminals, indicates the complexityof the integrative processing of information that occurs within theintrinsic cardiac nervous system. Thus, the interdependent activitydisplayed by populations of neurons in different regions of oneintrinsic cardiac ganglionated plexus, responding as they do to multiplecardiac sensory inputs, forms the basis for integrated regional cardiaccontrol.

[0074] Recent anatomical and functional data indicate the presence ofthe multiple neuronal subtypes within intrathoracic extracardiac andintrinsic cardiac ganglia. Within this neuronal hierarchy, the intrinsiccardiac nervous system functions as a distributive processor at thelevel of the target organ. The redundancy of function and non-coupledbehavior displayed by neurons in intrathoracic extracardiac andintrinsic cardiac ganglia minimizes the dependency for such control on asingle population of peripheral autonomic neurons. In this regard,network interactions that occur within the intrinsic cardiac nervoussystem to integrate parasympathetic and sympathetic efferent outflow tothe heart do so in coordination with intrathoracic extracardiac neuronsthat process afferent information from multiple sites in the heartduring each cardiac cycle. As no consistent coherence of activitygenerated has been identified among neurons in intrinsic cardiac andintrathoracic extracardiac ganglia, different populations of neurons,distributed spatially within the intrathoracic cardiac nervous system,respond to cardiac perturbations in a coordinate fashion. If neurons inone part of this neuronal network respond solely to inputs from a singleregion of the heart, such as the mechanosensory neurites associated witha right ventricular ventral papillary muscle, then the potential forimbalance within the different populations of neurons regulating variouscardiac regions might occur. A relatively low level of inputs on aspatial scale to the intrinsic cardiac nervous system would result inlow coherence among its components. In contrast, excessive input to thisspatially distributed nervous system would destabilize it, leading tocardiac arrhythmia formation, etc.

[0075] Regional control of cardiac function is dependent upon thecoordination of activity generated by neurons within intrathoracicautonomic ganglia and the central nervous system. The hierarchy ofnested feedback loops therein provides precise beat-to-beat control ofregional cardiac function. Contrary to classical teaching, intrathoracicautonomic ganglia act as more than simple relay stations for autonomicefferent neuronal control of the heart. Within the hierarchy ofintrathoracic ganglia and nerve interconnections, complex processingtakes place that involves spatial and temporal summation of sensoryinputs, preganglionic inputs from central neurons and intrathoracicganglionic reflexes activated by local cardiopulmonary sensory inputs.The activity of neurons within intrathoracic autonomic ganglia islikewise modulated by circulating hormones, chief among them beingcirculating catecholamines and angiotensin II.

[0076] The progressive development of cardiac disease is associated withmaladaptation of these neurohumoral control mechanisms. Differencesexist in autonomic control of the heart before any overt cardiovasculardisease occurs and such differences critically influence the outcome atthe time of ischemic heart disease onset. Differential remodeling of thecardiac neuron hierarchy (central and peripheral) for reflex control ofthe heart occurs in animals susceptible verses resistant to developmentof ventricular fibrillation during the evolution of chronic myocardialischemia/infarction. Understanding neuronal reorganization/remodelingthat occurs within the peripheral autonomic nervous system and theinteractions that occur between this neural remodeling and theremodeling of the myocardium leads to the novel approaches as presentlydisclosed and claimed for anti-arrhythmia therapy and also to therapiesdirected at ischemic heart disease and protection of the heart.

[0077] With respect to neural control of the heart, the intrathoracicganglia and their interconnections form the final common pathway forautonomic modulation of cardiac function. Data summarized and presentedherein indicates in support of the presently claimed and disclosedinvention that intrathoracic autonomic ganglia contain a heterogeneouspopulation of cell types including afferent, efferent and local circuitneurons. Yet, as a group, the intrathoracic reflexes mediated withinthese peripheral autonomic ganglia function in a coordinated fashionwith central neurons located in the spinal cord, brainstem andsupraspinal regions to regulate cardiac output on a beat to beat basis.

AFFERENT NEURONS

[0078] Cardiac afferent neurons. Sensory afferent neurons provide theautonomic nervous system with information about blood pressure, bloodvolume, blood gases as well as the mechanical and chemical milieu of theheart. For sensory inputs from cardiopulmonary regions, the nodose anddorsal root ganglia are classically recognized as providing sensoryinputs to the brainstem and spinal cord respectively. Data indicatesthat intrathoracic extracardiac (i.e. stellate and middle cervicalganglia) and intrinsic cardiac ganglia also contain afferent neuronswhose sensory neurites lie variously within the heart, lungs and greatthoracic vessels. Additional sensory inputs for the control of cardiacautonomic neurons arise from baroreceptors and chemoreceptors locatedalong the aortic arch, carotid sinus and carotid bodies as well as fromother afferent neural elements within the CNS, especially thehypothalamus.

[0079] Nodose Ganglia Afferent Neurons. The nodose receive cardiacafferent inputs from sensory neurites located in atrial and ventriculartissues. These sensory neurites preferentially sense chemical stimuli,with a few responding to mechanical stimuli or both modalities. Theresponse characteristics to induced stimuli are likewise divergent withmechanical stimuli exerting short-lived effects, while the augmentationin activity elicited by chemical stimuli far outlast the appliedstimulus. While inputs from these receptors contribute to overallcardiovascular regulation, they are not normally perceived.

[0080] Dorsal Root Ganglia (DRG) Afferent Neurons. The cell bodies ofDRG afferent neurons, receiving input from cardiac sensory neurites, arelocated in C₆-T₆ dorsal root ganglia. The sensory neurites of most ofthese afferent neurons transduce chemical and mechanical stimuli. Theinputs from this subpopulation of cardiac afferent neurons subservenormal cardiovascular regulation, as well as nociception whenexcessively activated.

[0081] Intrathoracic Afferent Neurons. Functional and anatomical dataindicate that intrathoracic autonomic ganglia contain afferent soma. Thesensory neurites associated with these afferent neurons are variouslylocated in atrial, ventricular, major vascular and pulmonary tissues.Most are responsive to mechanical and chemical stimuli. These afferentneurons continue to influence intrathoracic efferent postganglionicoutflows to the heart even after long-term decentralization ofintrathoracic ganglia. Such intrathoracic afferent neurons provideinputs to the intrathoracic short-loop feedback control circuits thatinvolve intrinsic cardiac and intrathoracic extracardiac neurons. Theseintrathoracic neural circuits, acting in concert with CNS mediatedreflexes, dynamically control regional cardiac function throughout eachcardiac cycle to maintain electrical stability of the heart and protectthe myocytes.

[0082] Aortic and Carotid Artery Baroreflexes. Stretch receptors,sensitive to changes in vessel size, are found on thoracic and cervicalarteries, being concentrated on the aortic arch and the carotid sinus.They provide inputs to neurons within the medulla and spinal cordproportional to systemic arterial blood pressure. Inputs from thesesensory neurites course centrally in the IX and X cranial nerves tosynapse with neurons located in the nucleus of the medullary solitarytract. Via multi-synaptic connections, these afferent inputs modulatethe activity of cardiac parasympathetic efferent preganglionic neuronslocated primarily in the nucleus ambiguus. They also influencesympathetic efferent neuronal outflow to the heart via brainstemprojections to the intermediolateral (IML) region of the spinal cord.The baroreflex so involved represents a negative feedback system thatmodulates cardiac function and peripheral vascular tone in response toeveryday stressors.

EFFERENT NEURONS

[0083] Sympathetic efferent neurons. The somata of sympatheticpreganglionic efferent preganglionic neurons which regulate the heartare located within the intermediolateral (IML) cell column of the spinalcord, projecting axons via the rami T1-T5 to synapse with sympatheticpostganglionic neurons contained within various intrathoracicextracardiac and intrinsic cardiac ganglia. Activation of thesesympathetic efferent projections augments heart rate, changes patternsand speed of impulse conduction through the electrical system of theheart and increases contractile force in atrial and ventricular tissues.Sympathetic efferent postganglionic somata that project axons to variouscardiac effector tissues are localized in intrathoracic extracardiac andintrinsic cardiac ganglia. Classically, the somata of sympatheticefferent postganglionic neurons that innervate the heart have beenthought to be restricted to the stellate ganglia. However, cardiacsympathetic efferent postganglionic soma have also been identified inthoracic middle cervical, mediastinal and intrinsic cardiac ganglia. Asubpopulation of intrinsic cardiac neurons express the catecholaminergicphenotype, these neurons thus contain the necessary enzymes to convertL-DOPA to dopamine and norepinephrine. The intrinsic cardiac nervoussystem also contains a separate population of small intenselyfluorescent (SIF) cells that display tyrosine hydrolyaseimmunoreactivity. Some of these project to adjacent principal intrinsiccardiac neurons.

[0084] Parasympathetic efferent neurons. The somata of cardiacparasympathetic efferent preganglionic neurons within the brainstem arelocated primarily within the nucleus ambiguous, with lesser numbersbeing located in the dorsal motor nucleus and regions in between. Axonsfrom these preganglionic soma project via the X cranial nerve to synapsewith parasympathetic efferent postganglionic neurons located withinvarious intrinsic cardiac ganglia (see hereinafter below). Activation ofparasympathetic efferent neurons depresses heart rate, slows the speedof impulse conduction through the heart, induces major suppression ofatrial muscle contractile force and evokes negative inotropic effects onventricular contractile force.

LOCAL CIRCUIT NEURONS

[0085] A subpopulation of neurons contained within extracardiac andintrinsic cardiac intrathoracic autonomic ganglia function tointerconnect neurons within individual ganglia and between neurons inseparate intrathoracic ganglia; these are called local circuit neurons.Preliminary data indicate that these neurons are involved in processingof afferent information to coordinate sympathetic and parasympatheticefferent outflows to cardiac effector sites. Interactions within thisneuron population form the substrate for generation of the basalactivity within peripheral autonomic ganglia, especially whenintrathoracic ganglia are disconnected from the influence of centralneurons.

ORGANIZATION OF THE INTRINSIC CARDIAC NERVOUS SYSTEM

[0086] The cardiac nervous system consists of distinct ganglia clustersthat function in an interdependent manner to modulate regional cardiacfunction. To date, eight separate ganglia clusters have been identifiedwithin the canine intrinsic nervous system, five associated with atrialtissues and three with ventricular tissue.

[0087] The five atrial ganglionated plexuses include: 1) the rightatrial ganglionated plexus localized in fatty tissue on the ventralsurface of the common right pulmonary vein complex; 2) the inferior venacava-inferior atrial ganglionated plexus located on the inferior rightatrium adjacent to the inferior vena cava; 3) the dorsal atrialganglionated plexus located on the dorsal surface of the atria betweenthe common pulmonary veins, immediately caudal to the right pulmonaryartery; 4) the ventral left atrial ganglionated plexus contained withinfat on the caudal-ventral aspect of the left atrium adjacent to the AVgroove; and 5) the posterior atrial ganglionated plexus.

[0088] The three major ventricular ganglionated plexi are: 1) the rightlateral ventricular ganglionated plexus located adjacent to the originof the right marginal artery; 2) the left lateral ventricularganglionated plexus located adjacent to the origin of the left marginalartery; and 3) the cranial medial ventricular ganglionated plexuslocated in fatty tissues surrounding the base of the aorta and mainpulmonary artery. Of these eight clusters of ganglia, functions havebeen primarily ascribed to five of them: neurons in the right atrial andposterior atrial ganglionated plexus have been shown to exertpreferential control over the sinoatrial node; those in inferior venacava-inferior atrial ganglia exert predominant control over inferioratrial and atrioventricular conductile tissues. Neurons in dorsal atrialand cranial medial ventricular ganglia are principal modulators ofcontractile tissue.

NEUROHUMORAL INTERACTIONS CONTRIBUTING TO CARDIAC CONTROL

[0089]FIG. 1 is a graphical representation of the neurohumoralinteractions involved in control of cardiac function. Data indicatesthat a hierarchy of peripheral autonomic neurons functioninterdependently via nested feedback loops to regulate cardiac functionon a beat-to-beat basis. FIG. 1, therefore, summarizes the concept ofneural control of the heart as mediated by intrathoracic extracardiacand intracardiac neurons which are continuously influenced by descendingprojections from higher centers in the spinal cord, brainstem, andsuprabulbar regions. Each successive synaptic relay point within thisautonomic outflow, from the brainstem to the heart, is in turninfluenced by afferent feedback from various cardiopulmonary andvascular afferent receptors. Accumulating evidence suggests that theremay be at least four functionally distinct neuronal types within theintrinsic cardiac nerve plexus; parasympathetic postganglionic efferentneurons, local circuit neurons, adrenergic postganglionic efferentneurons and afferent neurons. Local circuit and cardiac afferent neuronsalso lie within intrathoracic extracardiac ganglia, along with thesympathetic postganglionic neurons.

[0090] With respect to intrathoracic autonomic ganglia, cholinergic andadrenergic efferent neurons in these ganglia represent the outputelements that project axons to cardiac electrical and mechanicaltissues. Local circuit neurons interconnect adjacent neurons within oneganglion or link neurons in separate clusters of intrathoracic ganglia.These interneurons are involved in coordination of neuronal activitywithin these peripheral autonomic ganglia, thereby providing theunderlying inputs necessary for the maintenance of basal autonomicneuronal discharge. Intrathoracic afferent neurons providemechanosensitive and chemosensitive inputs from cardiopulmonary regionsdirectly to intrinsic cardiac and extracardiac neurons, forming thebasis of the intrathoracic neural feedback system. Superimposed onactivities generated by neurons in peripheral autonomic ganglia areefferent inputs from preganglionic neurons in the brainstem and spinalcord that together exert tonic influences on regional cardiac tone. CNSpreganglionic inputs are, in turn, influenced by inputs from highercenters in the central nervous system and by afferent feedback fromcentral and peripheral sensory afferent neurons.

INTERACTIONS AMONG PERIPHERAL AUTONOMIC NEURONS

[0091] Cardiac performance is modulated by both sympathetic andparasympathetic efferent neuronal inputs. The induced change in anyregional cardiac function ultimately depends upon the intrinsiccharacteristics of the cardiac end-effector being innervated, the levelof efferent activity from the CNS to the periphery and interactionsoccurring within peripheral autonomic ganglia and at the respectivecardiac end-effectors.

[0092] Interactions at the organ level Anatomical and functional studiesindicate that sympathetic and parasympathetic efferent postganglionicnerve endings lie in close proximity to each other in the targettissues. Interactions among sympathetic and parasympathetic efferentprojections to the heart involve pre- and postjunctional mechanisms atthe end-effectors in cardiac tissue. Post-junctional interactionsinvolve differential modulation of adenylate cyclase via G-proteincoupled receptor systems (FIG. 1). Catecholamines, released fromsympathetic efferent projections or derived from the circulation,influence myocardial tissues by binding primarily to β₁ andβ₂-adrenoceptors. Myocardial β adrenergic receptors are coupled to andstimulate adenylate cyclase via stimulatory guanine nucleotide bindingprotein (G_(s)). Acetylcholine, released from parasympathetic efferentpostganglionic neurons, binds to cardiomyocyte M₂ muscarinic receptorswhich, in turn, are coupled to and inhibit adenylate cyclase viainhibitory guanine nucleotide binding protein (G_(i)). The interactionsbetween these two receptor-coupled systems at the adenylate cyclaselevel ultimately determine the rate of formation of cAMP and therebymyocyte second messenger function. The neural interactions that occur atcardiac end-effectors involve primarily modulation of neurotransmitterrelease from pre-junctional synaptic terminals. Neural release of theprincipal mediators norepinephrine and acetylcholine, along with theco-release of various neuropeptides (e.g. NPY and VIP) act on specificreceptors associated with sympathetic or parasympathetic efferent axonterminals. These mechanisms act to modulate subsequent neurotransmitterrelease.

[0093] Interactions within the ICN. Various lines of evidence indicatethat peripheral sites that are separate from the end-effectorscontribute to mediating sympathetic-parasympathetic interactions for thecontrol of regional cardiac function. Stimulating parasympathetic and/orsympathetic efferent projections to the heart activates subpopulationsof intrinsic cardiac neurons. These extrinsic autonomic projectionsconverge on separate aggregates of intrinsic cardiac neurons, each ofwhich exhibit preferential control over regional cardiac function. Withrespect to control of chronotropic function, surgical disruption of theright atrial ganglionated plexus eliminates direct vagal projections tothe sinoatrial node. Sympathetic efferent neuronal control ofchronotropic function and the vagal inhibition of the sinus tachycardiaproduced by cardiac sympathetic efferent neurons are maintained. Theseresidual sympathetic-parasympathetic efferent neuronal interactionsoccur at the level of the heart and are prejunctional to the sinoatrialnode. As shown herein, these residual interactions occur within theintrinsic cardiac nervous system. Whether such intraganglionic autonomicinteractions play correspondingly roles in modulation of dromotropic andinotropic function has yet to be determined.

[0094] Intraganglionic interactions within the intrinsic cardiac nervoussystem depend in large part on common shared afferent inputs and/orinterconnections mediated via local circuit neurons. In order toevaluate these interactions, separate populations of neurons wererecorded in the ventral right atrial ganglionated plexus (RAGP) in basalstates and during discrete mechanical and chemical stimuli ofventricular neurites. In basal states, the coherence of activitygenerated by the two populations of RAGP neurons fluctuated with aperiodicity of 30-50 s and with an average peak coherence of 0.88±0.03.Coherence was increased in conjunction with the enhanced neuronalactivity evoked during exposure of ventricular sensory inputs tomechanical and chemical (nitroprusside, veratridine, bradykinin,adrenergic agonists or glutamate) stimuli. The interdependent activitydisplayed by the population of neurons in different regions of oneintrinsic cardiac ganglionated plexus, depending as they do on multiplecardiac sensory inputs, forms the basis for coordination of regionalcardiac function within the intrinsic cardiac nervous system.

[0095] Interactions within the intrathoracic nervous system.Coordination of autonomic outflows from intrathoracic neurons tocardiomyocytes depends to a large extent on sharing of inputs fromhigher centers along with interactions among and between variousperipheral ganglia. Interactions within and between intrathoracicganglia involve local circuit neurons (see herein above). Activitiesgenerated by neurons in intrinsic cardiac ganglia demonstrate noconsistent short-term relationship to neurons in extracardiac ganglia.However, the sharing of cardiopulmonary afferent information actingthrough both intrathoracic and brain stem/spinal cord feedback loopspermits an overall coordination of effector control. Together, thesenested feedback control systems allow for a redundancy in neural controlof the heart while at the same time maintaining the flexibility todifferentially modulate regional cardiac function.

ELECTROPHYSIOLOGY OF INTRINSIC CARDIAC GANGLIA

[0096] In Vivo Studies. Cardiac neurons generate spontaneous activity insitu, frequently exhibiting activity that is temporally related to thecardiac or respiratory cycles. Of the neurons that displayedcardiac-related activity, many are affected by mechano- or chemosensoryinputs from the heart. Trains of electrical stimuli delivered to axonsin the T1-T5 ventral roots activate a substantial population of stellateand middle cervical neurons. These data indicate a convergence ofpreganglionic inputs onto the extracardiac postganglionic soma,reflective of a functional amplification of such sensory input. Incontrast, trains of electrical stimuli delivered to the vagosympathetictrunks or stellate ganglia activate a much smaller population ofintrinsic cardiac neurons. Moreover, few intrinsic cardiac neurons areactivated after a fixed latency when extracardiac efferent neurons thatinnervate the heart are stimulated electrically, a finding indicative ofmonosynaptic interconnections to such neurons. These data indicate that,in contradistinction to extracardiac ganglia, substantial spatial andtemporal summation of inputs are required to modify the activitygenerated by neurons on the heart. Intrinsic cardiac neurons generatelow level activity in such a state consistent with a nerve network thatfunctions as a “low pass filter”, thereby minimizing the potential forimbalances within autonomic efferent neuronal inputs to the heart, aprocess which by itself could be arrhythmogenic.

[0097] In Vitro Studies. Intrinsic cardiac ganglia contain aheterogeneous population of neurons. An intracellular recording fromisolated whole mount aggregates of intrinsic cardiac ganglia indicatesthat complex neural interactions occur within the heart. Studies onaggregates of intrinsic cardiac ganglia derived from different speciesfurther indicate that the resting membrane potentials of these neuronsis approximately −60 mV, with relatively low input resistances andthresholds for the generation of action potential being approximately 20mV more positive than the resting membrane potential. These propertiesare consistent with neurons functioning with low excitability. Noevidence for ramp-like pacemaker activities has been found withinmammalian intrinsic cardiac neurons in vitro. Thus spontaneous activitygenerated by such neurons in vivo likely reflects underlying cell-cellinteractions. For orthodromic stimulation there is substantialdispersion in time of the evoked excitatory postsynaptic potentials(EPSP's) generated by a given intrinsic cardiac neuron, indicative ofpolysynaptic inputs to neurons within the intrinsic cardiac nervoussystem. After the generation of action potentials, prolongedafterhyperpolarizations are produced by these cells, an additionalfactor which limits the excitability of intrinsic cardiac neurons insitu. Chronic disruptions of nerve inputs to these ganglia evokeschanges in membrane properties which may result in increasedexcitability within the ganglionated plexus.

[0098] Intracellular recordings from isolated aggregates of intrinsiccardiac ganglia have identified both cholinergic and non-cholinergicsynaptic mechanisms coexisting within intrinsic cardiac ganglia. In ratsand pigs only fast excitatory postsynaptic potentials are displayed byintrinsic cardiac neurons in response to orthodromic stimulation ofclosely adjacent intraganglionic axons. These postsynaptic potentialsare substantially attenuated, but not completely eliminated, bynicotinic cholinergic blockade. In the dog, orthodromic stimulation ofpresynaptic fibers in these nerves elicits fast and slow postsynapticpotentials within intrinsic cardiac neurons. Fast excitatorypostsynaptic potentials are mediated by cholinergic nicotinic receptors,while the slow excitatory and slow inhibitory potentials are mediated bycholinergic muscarinic receptors. In the pig direct application ofnorepinephrine modifies the properties of about 25% of identifiedintrinsic cardiac neurons. These data indicate that intrinsic cardiacneurons possess muscarinic cholinergic, nicotinic cholinergic as well asadrenergic receptors. As detailed hereinafter, many other putativeneurotransmitters likewise modify electrical events of intrinsic cardiacneurons. These neurochemicals may play important roles in the modulationof intrinsic cardiac neuronal activity.

[0099] In summary, intrathoracic autonomic ganglia do not function asobligatory synaptic stations for autonomic efferent neuronal input tothe heart. Instead, they are capable of complex signal integrationinvolving afferent, local circuit as well as parasympathetic andsympathetic efferent neurons. While the physiological properties ofextracardiac autonomic ganglia tend to amplify CNS and afferent feedbackinputs, those of the intrinsic cardiac nervous system act to limitcardiac excitability. As such, the final common pathway of cardiaccontrol—the intrinsic cardiac nervous system—appears to function as a“low pass” filter to minimize transient neuronal imbalances arising fromseparate sympathetic and parasympathetic efferent neuronal inputs to theheart. In conjunction with this local afferent feedback mechanism,neurons in intrathoracic ganglia also mediate local cardio-cardiacreflexes at sites separate from those on the heart and the CNS. Thesynaptic events underlying such intraganglionic interactions involvemultiple neurotransmitters that interact with various neuronal receptorsto exert rapid acting neuronal membrane conductance and/or longer-termmodulation of synapses within the intrinsic cardiac nervous system.

SYNAPTIC MECHANISMS ASSOCIATED WITH NEURONS IN INTRATHORACIC AUTONOMICGANGLIA

[0100] Cholinergic Mechanisms. Synaptic transmission in autonomicganglia principally involves the release of acetylcholine by presynapticterminals and subsequent binding of that neurotransmitter to nicotiniccholinergic receptors on postganglionic neurons. In mammals thissynaptic junction is not obligatory, indicating that a significantconvergence of inputs may be necessary to evoke postganglionic neuronalactivity. Thus the potential for synaptic integration exists withinintrathoracic autonomic ganglia. Nicotinic and muscarinic cholinergicreceptors have been associated with intrathoracic autonomic neurons.Furthermore, blockade of nicotinic receptors attenuates, but does noteliminate, activity generated by the intrinsic cardiac neurons.Muscarinic blockade attenuates excitatory and inhibitory synapticfunction within intrinsic cardiac ganglia as well. These sets of dataindicate that acetylcholine exerts both mediator and modulator effectsat synapses within intrathoracic autonomic ganglia.

[0101] Application of nicotine to intrathoracic autonomic neurons canalter their activity and induce concomitant changes in regional cardiacfunction, whether the neurons are located in extracardiac or intrinsiccardiac ganglia. Nicotinic activation of intrinsic cardiac neuronsevokes a biphasic cardiac response, with initial suppression in regionalcardiac function being followed by augmentation. Acute decentralizationof intrathoracic ganglia from the CNS attenuates, but does noteliminate, such effects. In time, following chronic decentralization ofintrathoracic ganglia including those on the heart as with cardiactransplantation, peripheral nerve networks remodel to sustain cardiacfunction. For cholinergic receptor systems, the remodeling primarilyinvolves augmentation of excitatory influences mediated by muscarinicreceptors.

[0102] Non-cholinergic Mechanisms. Blockade of nicotinic cholinergicreceptors attenuates, but does not eliminate, the activity generated byneurons within the intrathoracic autonomic ganglia. These data indicatethat non-nicotinic putative neurotransmitters act as mediators forsynaptic transmission within the intrathoracic neuronal system.Anatomical and physiological studies have identified multiple putativeneurotransmitters in association with the mammalian intrinsic cardiacganglia which include purinergic agonists, catecholamines, angiotensinII, calcitonin gene-related peptide, neuropeptide Y, substance P,neurokinins, endothelin and vasoactive intestinal peptide. Many of theseputative neurochemicals arise from neurons whose cell bodies are locatedin stellate, middle cervical or mediastinal ganglia, while others may besynthesized by neurons intrinsic to the heart. Direct application ofvarious neurotransmitters adjacent to neurons in intrinsic cardiacganglia modifies the activities they generated, often resulting inconcomitant changes in cardiac pacemaker and/or contractile behavior.

[0103] Intrinsic cardiac ganglia contain a heterogeneous population ofneurons that utilize cholinergic and non-cholinergic synapses to controlintraganglionic, interganglionic and nerve effector organ cellactivities. Some of these neurotransmitters subserve short durationsynaptic actions (e.g. acetylcholine) while others modulate pre- and/orpost-synaptic function over longer periods of time (e.g., neuropeptideY).

NEURAL REMODELING IN THE HEART ASSOCIATED WITH MYOCARDIAL ISCHEMIA

[0104] Myocardial ischemia and infarction can induce substantial changesin the intrathoracic nerve networks and their reflex control of regionalcardiac function. Chen and co-workers [(41;42;122)] have recentlyproposed the sprouting hypothesis of sudden cardiac death: namely,“Myocardial Ischemia results in nerve injury, followed by sympatheticnerve sprouting and regional myocardial hyperinnervation. The couplingbetween augmented sympathetic nerve sprouting with electrical remodeledmyocardium results in VT, VF and SCD.” The results of these studies andothers have indicated that the evolution of cardiac pathologies may beassociated with a heterogeneous distribution of efferent projections tocardiac end-effectors. Myocardial ischemia may also alter theneurochemical profile of that innervation; e.g. expression of vasoactiveintestinal polypeptide and calcitonin gene-related peptide are enhancedin sympathetic neurons after myocardial infarction. Finally, theevolution of cardiac pathology can be associated with disruptions of theintrinsic cardiac nervous system and its ability to process afferentinformation. Such changes compromise the abilities of the peripheralnerve networks to maintain homogeneity for reflex control of regionalcardiac function. This neural remodeling, when coupled with theischemic-induced heterogeneous electrical remodeling of cardiacmyocytes, creates a synergistic substrate for arrhythmias and suddencardiac death.

INTERACTIONS BETWEEN CNS AND INTRATHORACIC NEURONAL NETWORKS:IMPLICATIONS FOR TREATMENT OF MYOCARDIAL ISCHEMIA AND ANGINA PECTORIS

[0105] Myocardial ischemia reflects an imbalance in the supply:demandbalance within the heart with resultant activation of cardiac afferentneurons and, as a consequence, the perception of symptoms (i.e., anginapectoris). In addition to such nociceptive responses, activating cardiacafferent neurons can elicit autonomic and somatic reflexes.Pharmacological, surgical and angioplasty therapies are commonly used toimprove symptoms and cardiac function in patients exhibiting anginapectoris. While these treatments are usually successful, some patientsstill suffer from cardiac pain following these procedures. Recently,epidural stimulation of the spinal cord (SCS or Dorsal Cord Activation,DCA) has been suggested as an alternative to bypass surgery in high-riskpatients. With DCA, high frequency, low intensity electrical stimuli aredelivered to the dorsal aspect of the T1-T3 segments of the thoracicspinal cord. This therapy decreases the frequency and intensity ofanginal episodes. DCA reduces the magnitude and duration of ST segmentalteration during exercise stress in patients with cardiac disease,improves myocardial lactate metabolism and increases workload tolerance.The mechanisms whereby this mode of therapy produces such beneficialeffects are, to date, poorly understood an although used extensively inEurope, are not a standard of practice within the United States.

[0106] Since intrathoracic cardiac neurons have been found to playimportant modulatory roles in cardiac regulation, the use of DCA and itseffects on the activity generated by intrinsic cardiac neurons has beenstudied and is at least one component of the presently claimed anddisclosed invention. Transient cardiac ventricular ischemia increasesthe activities generated by intrathoracic ganglia, including those onthe heart. Excessive focal activation of intrathoracic neural circuitscan induce cardiac dysrhythmias, even in normally perfused hearts. DCAresults in an immediate suppression in intrinsic cardiac neuronalactivity. A neuro-suppressor effect imposed in the intrinsic cardiacnervous system occurs whether DCA is applied immediately before, duringor after coronary artery occlusion (FIGS. 2 and 3). Furthermore, thesuppression of intrinsic cardiac neuronal activity persists even aftercessation of DCA (FIG. 4). That transection of the ansae subclaviaeliminated these effects indicates that they primarily involve thesympathetic nervous system.

[0107] The synaptic mechanisms and specific pathways mediating theseresponses likely involve both sympathetic afferent and efferent neurons.Dorsal cord activation excites sensory afferent fibers antidromicallysuch that endorphins or neuropeptides such as calcitonin gene-relatedpeptide or substance P are locally released in the intrinsic cardiacganglia and myocardium. Opiates and neuropeptides can also influenceintrinsic cardiac neurons (see herein-above). Spinal cord stimulationalso suppresses intrinsic cardiac adrenergic as well as local circuitneurons as the result of altered sympathetic efferent preganglionicneuronal activity. It is also known that activation of sympatheticefferent preganglionic axons suppresses many intrathoracic reflexes thatare involved in cardiac regulation. Thus these neuro-suppressor effectsappear to be due, in part, to activation of inhibitory synapses withinintrathoracic ganglia. Recent clinical experience with DCA highlightsthe dynamic interactions that can occur between central andintrathoracic neurons, demonstrating the potential for effectiveclinical treatment of cardiac pathology via modulation of theintrathoracic nervous system or the intrinsic cardiac nervous system.

COORDINATION OF ACTIVITIES WITHIN AND BETWEEN GANGLIA OF THE INTRINSICCARDIAC NERVOUS SYSTEM

[0108] FIGS. 2-4 summarize the induced changes in intrinsic cardiacnerve activity produced by transient coronary artery occlusion (CAO) andtheir modulation by descending projections from the T1-T3 segments ofthe spinal cord. Note the augmentation in activity within the atrial andventricular neurons (FIG. 2) produced by CAO is attenuated by electricalstimulation of the dorsal aspects of the T1-T3 segments of the spinalcord (FIG. 3, DCA; Dorsal Cord Activation). The suppression of activityinduced by DCA on the intrinsic cardiac neuronal activity is maintainedlong after the termination of spinal cord stimulation (FIG. 4).

[0109] As shown in FIG. 5, activity generated by two differentpopulations of intrinsic cardiac neurons contained within the rightatrial ganglionated plexus. Arrow indicates application of veratridineto the epicardium of the left ventricle. At baseline, note the cyclingof activity with a periodicity of 20 seconds. In the unstressedcondition, this bursting is usually associated with increasedcoordination of activity between the two populations of neurons (seebottom trace). When an afferent stress is imposed to the ICN, as withapplication of epicardial veratridine, activity increased in both sitesand the coherence of activity generated by these two populations ofneurons approached unity.

FUNCTIONAL REMODELING OF THE INTRINSIC CARDIAC NERVOUS SYSTEM INRESPONSE TO CHRONIC MYOCARDIAL ISCHEMIA

[0110]FIGS. 6 and 7 summarize the changes induced in baselineelectrophysiology and in the neural control of cardiac electricalfunction in response to chronic myocardial ischemia produced by chronicplacement of an ameroid constrictor on the left circumflex artery. Thisconstrictor produces a progressive occlusion of the artery withinduction of collateral blood vessels and does not produce musclenecrosis or scar formation.

DIFFERENTIAL CONTROL OF NEUROTRANSMITTER RELEASE WITHIN THE CARDIACINTERSTITIUM

[0111] Exogenous administration of ANG II into the blood supply for theright atrial ganglionated plexus increased NE concentration in thecardiac interstitial fluid (ISF) to the same extent as achieved duringelectrical stimulation of the stellate ganglia (FIG. 8A) in theanesthetized dog. LV dP/dt correlated with ISF NE release. However, NEspillover into the coronary sinus occurred only during sympatheticefferent neuronal stimulation (FIG. 8C). ISF EPI levels increasedmoderately with stellate stimulation and to levels equal to NE releasewith ANG II stimulation. This differential release of catecholaminesfrom cardiac nerves occurred in spite of a 40-fold higher NE compared toEPI content in the dog LV myocardium (237±33 vs. 6.4±1.0 ng/g). Neitherstellate nor ANG II stimulation evoked EPI spillover into the coronarysinus. Dorsal cord activation (FIG. 8B) evoked a release of EPI into theISF equivalent to stellate stimulation, but with only a modest increasein ISF NE. These data illustrate the potential for differential neuralrelease of catecholamines within the heart depending on how efferentoutflows are activated and underscore the importance of simultaneousmeasurements of ISF and transcardiac release in evaluation of the neuralcontrol of regional cardiac function.

ELECTROPHYSIOLOGICAL PROPERTIES OF IN VITRO CARDIAC GANGLIA

[0112] Disruption of nerve projections to or within the intrinsiccardiac nervous (ICN) system are associated with alterations in thepassive and active properties of the cardiac neurons. chronicinterruption of the extrinsic nerve inputs to the ICN has been shown toproduce changes in membrane properties that lead to increased networkexcitability within this ganglionated plexus. Intrinsic cardiac neuronsremain responsive to cholinergic synaptic inputs. The cholinergicreceptor systems are differentially affected by disruption of nerveinputs to the ICN, with muscarinic responsiveness being enhanced (FIG.9). Non-cholinergic neurotransmitters can modulate the activity of theseneurons. FIG. 10 illustrates the interaction between acetylcholine andthe peptide, substance P.

QUANTIFICATION OF THE INNERVATION PROFILE FOR THE CANINE HEART

[0113] Data indicate that the progression of cardiac disease isassociated with myocyte and neural remodeling. The neural remodelinglikely includes degenerative and regenerative aspects. The net result isthe potential for heterogeneous innervation to various regions of theheart. Chen et al. have therefore proposed the “nerve sproutinghypothesis of sudden cardiac death”. As illustrated in FIG. 11 by usingimmunohistochemical techniques, characterization of innervation density(panel B) and types of fibers (panel A) within ganglia and cardiactissues has been accomplished.

[0114] Interactions within the intrinsic cardiac nervous system dependin large part on common shared afferent inputs and/or interconnectionsmediated via local circuit neurons. The degree of coordination betweenaggregates of intrinsic cardiac neurons is influenced by proximity andthe activation state of afferent inputs. In basal states, the degree ofcoherence of activity within a single cardiac ganglia waxes and waneswith a periodicity of approximately 20-30 sec. In response to enhancedneuronal activity, evoked during activation of their associated sensoryinputs, that coherence increases. For neurons contained within differentintrinsic cardiac ganglia, lesser degrees of coherence of basal activitybetween them, but this coherence increases during stimulation ofafferent inputs owing to common shared inputs between them.

[0115] There are two distinct classes of sensory input affecting ICNactivity: a phasic input, whose influence is short-lived and subservesrapid feedback processes within the ICN and a dynamic input whoseinfluence is determined by the context/history of its activation andwhose influence on ICN activity is long-lived. Mechano-sensitiveneurites subserve the phasic inputs and chemo-sensitive neuritessubserve the neural “memory”.

[0116] Myocardial ischemia and infarction induce substantial changes inthe intrathoracic nerve networks and their reflex control of regionalcardiac function including protection and stabilization of electricalactivity of the heart. Chronic myocardial ischemia induces aheterogeneous distribution of efferent projections to cardiacend-effectors. Heterogeneous distribution of sympathetic fibers to theleft ventricle results in similar heterogeneous release ofcatecholamines into the interstitial space during stimulation of theefferent nerves. Myocardial ischemia alters the neurochemical profile ofthat innervation, with differential increases in neuropeptide contentwithin subsets of neurons contained within the intrinsic cardiac nervoussystem. The evolution of cardiac pathology is associated withdisruptions of the intrinsic cardiac nervous system and its ability toprocess afferent information and such changes are more evident in theCMVPG than the RAGP intrinsic cardiac ganglia. Animals that exhibitindices of higher vagal tone (higher baroreflex sensitivity and higherheart rate variability) demonstrate lesser degrees of ischemic-inducedneural remodeling.

[0117] DCA can exert long-term modulation of the activities within theintrinsic cardiac nervous system. While initial studies indicate thatcatecholamines are released in response to DCA, it is anticipated thatDCA also will activate sensory afferent fibers antidromically such thatendorphins or neuropeptides such as calcitonin gene-related peptide orsubstance P are locally released in the intrinsic cardiac ganglia andmyocardium. Opiates and neuropeptides can also influence the intrinsiccardiac neurons. DCA also suppresses intrinsic cardiac adrenergic aswell as local circuit neurons via altered sympathetic efferentpreganglionic neuronal input. Activation of sympathetic efferentpreganglionic axons suppresses many intrathoracic reflexes that areinvolved in cardiac regulation. Thus these neuro-suppressor effects maybe due, in part, to activation of inhibitory synapses within intrinsicganglia.

[0118] Heterogeneous alterations within the intrinsic cardiac ganglia orat the end-terminus of the autonomic innervation to the ischemicmyocardium are major contributors to the increased incidence of suddencardiac death in patients with coronary artery disease. Chronic DCAameliorates ischemia-induced remodeling within the intrinsic cardiacnervous system and thereby reduces the heterogeneous neural substratethat predisposes the susceptible animals to ventricular arrhythmias andsudden cardiac death.

[0119] Control of regional cardiac electrical and mechanical function isdependent upon varied neural inputs from intrathoracic autonomicganglia, the spinal cord and brainstem, as well as by circulatingneurohumoral agents. Neural control of the heart is dependent upon thecoordination of activity generated by neurons within intrathoracicautonomic ganglia and the CNS. The hierarchy of nested feedback loopstherein provides precise beat-to-beat control over regional cardiacfunction. Within the hierarchy of intrathoracic ganglia and nerveinterconnections, complex processing takes place that involves thesummation of preganglionic inputs from central neurons with thosederived from cardiopulmonary sensory inputs.

[0120] Excessive activation of the intrathoracic cardiac efferentnervous system can provoke cardiac arrhythmias, as can myocardialischemia. These maladaptations likely involve changes within the cardiacnervous system in addition to alterations in cardiomyocyte function.Differential adaptations of cardiomyocyte ion channels (e.g. IK and ICa)and intercellular connections during the progression of cardiac diseasehave been termed “electrical remodeling.” Recent data indicates thatneurohumoral control mechanisms likewise reorganize during progressioninto certain cardiac diseases and are referred to as “neurohumoralremodeling.”

[0121] Changes in autonomic outflow accompany and influence theprogression of cardiac disease. Sympathetic efferent neuronal activationcontributes to sudden cardiac death in patients with ischemic as well asnon-ischemic heart disease. The ATRAMI study demonstrates thatbaroreflex sensitivity and heart rate variability predict risk forcardiovascular mortality and myocardial infarction. Electricalstimulation of vagal efferent neurons suppresses the tendency toventricular fibrillation formation in dogs with depressed vagal reflexactivity as measured by baroreflex sensitivity. Yet, pharmacologicalagents that increase vagal efferent neuronal tone, such as a low-dosescopolamine, do not confer similar degrees of protection.

[0122] The mechanism(s) whereby activation of sympathetic efferentneurons and/or withdrawal of parasympathetic efferent neuronal toneincrease the risk for sudden death are not clear. However,post-infarction heterogeneous remodeling of cardiac innervation,including extracardiac sympathetic and intrinsic cardiac efferent neuralelements, likely contributes to the resultant cardiac electricalinstability. The present claimed and disclosed invention, as disclosedherein, outlines the evolution of neural remodeling associated withchronic myocardial ischemia and infarction and thus provides a steppingoff point for the development of treatments for cardiac pathologiesutilizing SCS or DCA.

[0123] After decades of progress, improvement in the management ofcardiac arrhythmias appears to have leveled off. The problem of suddencardiac death occurring as the result of an initial arrhythmic event hasnot been addressed (except perhaps through palliative public healthstrategies which include public access defibrillators, PAD). This stateof affairs is due, in part, to the fact that key pieces of informationregarding cardiac arrhythmia formation are still missing, including theability to identify the apparently normal individual at risk before anevent occurs. While changes in myocardial electrical events have beenwell characterized in the diseased heart, information concerning thecomplex neuronal organization regulating cardiac rhythm remains limited.The comprehensive presently claimed and disclosed invention whichincludes the knowledge of the complex processing which occurs within theintrathoracic nervous system, as well as between peripheral and centralcardiovascular neurons, provides a basis for understanding the role thatthe cardiac nervous system plays in regulating the electrical behaviorof not only the normal heart, but the diseased heart as well, thusproviding for novel therapeutic approaches for the effective treatmentof cardiac arrhythmias, sudden cardiac death or syncope of cardiacorigin by targeting discrete populations of neurons regulating regionalcardiac behavior.

[0124] Control of regional cardiac function is dependent upon propertiesintrinsic to cardiac electrical and mechanical tissues as modulated byneuronal reflexes arising at the level of the intrinsic cardiac andintrathoracic extracardiac nervous systems, in addition to well-knownspinal cord and brainstem reflexes. The proper function of this cardiacneuronal hierarchy is ultimately dependent on ongoing cardiovascularsensory and spinal cord neuronal inputs. The synergism of functionwithin the cardiac autonomic hierarchy and cardiac myocytes results in afinely balanced, rapidly responsive control system that is continuouslybeing upgraded to maintain adequate cardiac output. As outlinedhereinabove, the intrinsic cardiac ganglia form the principal finalcommon pathway for autonomic modulation of regional cardiac function.Maintenance of cardiac output depends not only on the Frank-Starlingmechanism and circulating catecholamines, but also on inputs from thisnervous system. Disruptions of the sensory inputs to the hierarchy ofautonomic neurons regulating the heart due to alterations in themechanical and/or chemical milieu of the heart can be associated withcompromised control of the heart.

ANATOMY AND FUNCTION OF THE INTRATHORACIC CARDIAC NERVOUS SYSTEM(INTRINSIC CARDIAC NERVOUS SYSTEM)

[0125] Divergent populations of cardiac neurons within differentintrathoracic ganglia interact on an ongoing basis to maintain adequatecardiac output, requiring little ongoing input from spinal cord neurons.neurons in this hierarchy interact to regulate normal cardiac functionon a beat-to-beat basis. The development of novel strategies to managecardiac disease necessitates not only a thorough understanding of theprocessing of information arising from cardiac and major intrathoracicvascular sensory neurites, but also inputs from central neurons. Neuronsin the spinal control exert preferential control over such intrathoracicneuronal processing of cardiac sensory information.

[0126] Human studies have shown that stimulation of the dorsal T1-T2segments of the spinal cord suppresses angina pectoris (sensoryinformation arising from the heart) without masking awareness of acutemyocardial ischemic episodes. The mechanisms whereby activation of thedorsal aspect of the cranial thoracic spinal cord produces improvedcardiac function and reduces symptoms of the ischemic myocardium are notcurrently understood. The experiments and resulting data from thepresently claimed and disclosed invention show that the anti-anginal andcardiac stabilization effects of such spinal cord modulation aremediated via stabilization of the intrathoracic nervous system,especially its intrinsic cardiac component. Neural control of cardiacfunction resides in the network of nested feedback loops made up of theintrinsic cardiac nervous system, extracardiac intrathoracic autonomicganglia, the spinal cord and brainstem. Within this hierarchy, theintrinsic cardiac nervous system functions as a distributive processorat the level of the target organ. The redundancy of function andnon-coupled behavior displayed by neurons in intrathoracic extracardiacand intrinsic cardiac ganglia minimizes the dependency for such controlon a single population of peripheral autonomic neurons. On the otherhand, network interactions occurring within the intrinsic cardiacnervous system integrate parasympathetic and sympathetic efferentoutflow with cardiovascular afferent feedback to modify cardiac rate andregional contractile force throughout each cardiac cycle. Thus, neuralcontrol of cardiac function resides in the network of nested feedbackloops made up of the intrinsic cardiac nervous system as well as theextracardiac intrathoracic nervous system, spinal cord and brainstem(FIG. 1).

[0127] The redundancy of function and non-coupled behavior displayed byneurons in intrathoracic extracardiac and intrinsic cardiac gangliaminimizes the dependency for such control on a single population ofperipheral autonomic neurons. Furthermore, network interactionsoccurring within the intrinsic cardiac nervous system integrateparasympathetic and sympathetic efferent outflow with afferent feedbackto modify cardiac rate and regional contractile force throughout eachcardiac cycle.

THE INTRINSIC CARDIAC NERVOUS SYSTEM

[0128] The intrinsic cardiac nervous system has been classicallyconsidered to contain only parasympathetic efferent postganglionicneurons that receive inputs from medullary parasympathetic efferentpreganglionic neurons. As such, intrinsic cardiac ganglia are viewed assimple relay stations and major autonomic neuronal control of the heartis purported to reside solely in the brainstem and spinal cord. However,current data indicates that centripetal as well as centrifugalprocessing occurs within the mammalian intrathoracic nervous system.This involves afferent neurons, local circuit neurons (i.e., neuronsthat interconnect neurons within one ganglion and neurons in differentintrathoracic ganglia), as well as sympathetic and parasympatheticefferent postganglionic neurons. The divergent populations of neuronswithin the intrinsic cardiac nervous are influenced by spinal cordneurons on an ongoing basis in the maintenance of adequate cardiacoutput. FIG. 1 provides an outline for the putative types of neurons andtheir interconnectivity within the cardiac neuronal hierarchy.

[0129] The development of novel therapeutic strategies to manageabnormal cardiac states necessitates a thorough understanding of notonly of the processing of information arising from sensory neurites invarious regions of the heart and great thoracic vessels, but how spinalcontrol neurons exert preferential control over the intrathoraciccardiac nervous system with particular reference to its target organ.Similarly, intrathoracic extracardiac sympathetic ganglia have beenthought to act solely as efferent relay stations for sympatheticefferent projections to the heart. However, recent anatomical andfunctional data indicate the presence of the multiple neuronal subtypeswithin the intrinsic cardiac nervous system. The intrathoracic nervoussystem, including its intrinsic cardiac component, is made up ofdifferent neuronal subtypes. These include afferent, local circuit aswell as adrenergic and cholinergic efferent postganglionic neurons.These neurons form the intrathoracic component of the central andperipheral neuronal feedback loops that regulate regional cardiodynamicson a beat-to-beat basis.

[0130] The intrinsic cardiac nervous system consists of multipleaggregates of neurons and associated neural interconnections, localizedto discrete atrial and ventricular regions. Among these distinctganglionated plexuses, preferential control of specific cardiacfunctions has been identified. For example, right atrial ganglionatedplexus (RAGP) neurons have been associated with primary, but notexclusive, control of SA nodal function and inferior vena cava-inferioratrial ganglionated plexus neurons primarily, but not exclusively, withcontrol of AV nodal function. One population of intrinsic cardiacneurons, the parasympathetic postganglionic ones, receives direct inputsfrom medullary parasympathetic preganglionic neurons. Anotherpopulation, adrenergic efferent ones, receives inputs from morecentrally located neurons in intrathoracic ganglia and the spinal cord.That ventricular sensory neurites continue to influence the activitygenerated by neurons on the heart following chronic decentralization ofthe intrinsic cardiac nervous system has been interpreted as indicatingthat the somata of afferent neurons are located within the intrinsiccardiac nervous system, some of which project axons to central neurons.This latter concept has received anatomical confirmation. Intrinsiclocal circuit neurons interconnect cardiac afferent to efferent neuronsthat innervate each region of the heart.

THE INTRATHORACIC EXTRACARDIAC NERVOUS SYSTEM

[0131] Neurons in intrathoracic ganglia, including those on the heart,receive constant inputs not only from spinal cord neurons, but also fromcardiac afferent neurons to modulate cardiac efferent neurons. Theactivity generated by most intrinsic cardiac neurons increases markedlyin the presence of focal ventricular ischemia. Furthermore, excessiveactivation of limited populations of intrinsic cardiac neurons inducescardiac dysrhythmias that lead to ventricular fibrillation, even innormally perfused hearts. Therapies that act to stabilize suchheterogeneous evoked activities within cardiac reflex control circuitshave obvious clinical importance. Proper information exchange among theintrathoracic components of the cardiac nervous system act in concert tostabilize the electrical and mechanical behavior of the heart,particularly in the presence of focal ventricular ischemia. Thus, use ofSCS or DCA is a means to stabilize the heart prior or post ischemia. Anobject of the present invention is to provide such treatmentmethodologies.

[0132] Consistent coherence of activity generated by differingpopulations of neurons is indicative of principal, direct synapticinterconnections between them or, conversely, the sharing by suchneurons of common inputs. Such relationships have been identified amongmedullary and spinal cord sympathetic efferent preganglionic neurons, aswell as among different populations of sympathetic efferentpreganglionic neurons. Different populations of neurons, distributedspatially within the intrathoracic cardiac nervous system, respond tocardiac perturbations in a coordinate fashion. If neurons in one part ofthis neuronal network respond to inputs from a single region of theheart, such as the mechanosensory neurites associated with a rightventricular ventral papillary muscle, then the potential for imbalancewithin the different populations of neurons regulating various cardiacregions might occur and, thus, its neurons would display littlecoherence of activity. In other words, relatively low levels of specificinputs on a spatial scale to the intrathoracic cardiac nervous systemwould result in low coherence among its various neuronal components. Onthe other hand, excessive input to this spatially distributed nervoussystem would destabilize it, leading to cardiac arrhythmia formation,etc.

INTERACTIONS AMONG INTRATHORACIC EXTRACARDIAC AND INTRISIC CARDIACNEURONS

[0133] One must know how neurons in intrinsic cardiac versusintrathoracic extracardiac ganglia interact to regulate regionalcontractile function in order to understand not only the complexity ofcardiac control, but also how the cardiac neuroaxis can be targetedtherapeutically to manage specific cardiac disease entities. Over thepast 30 years studies of the anatomy and function of the peripheralcardiac nervous system have taken place, focusing during the last decadeon its intrinsic cardiac component. The classical view of the autonomicnervous system presumes that its intrinsic cardiac component comprises aparasympathetic efferent neuronal relay station in which medullarypreganglionic neurons synapse with parasympathetic efferentpostganglionic neurons therein. In such a concept, the latter neuronsproject to end effectors on the heart with little or no integrativecapabilities occurring therein. Similarly, intrathoracic paravertebralganglia have been thought to represent synaptic stations for sympatheticefferent postganglionic neurons controlling the heart.

[0134] The intrinsic cardiac nervous system functions, according to thepresently claimed and disclosed invention, as a distributive processorat the level of the target organ. The redundancy of function andnon-coupled behavior displayed by neurons within intrathoracicextracardiac and intrinsic cardiac ganglia minimizes the dependency forsuch control on a single population of peripheral autonomic neurons. Inthat regard, network interactions occurring at the level of the heartintegrate parasympathetic and sympathetic efferent inputs with localafferent feedback to modify cardiac rate and regional contractile forcethroughout each cardiac cycle. A recent editorial by David Lathrop andPete Spooner of the NIH highlights the potential clinical relevance ofaltered processing of information by these populations of neurons suchthat a lack of coordination of data exchange within the cardiac neuronalaxis may lead to the genesis of cardiac arrhythmias. Hence theimportance of determining how neurons in intrathoracic extracardiac andintrinsic cardiac ganglia interact in the maintenance of adequatecardiac output.

[0135] The different populations of neurons distributed spatially withinthe intrathoracic cardiac nervous system respond to cardiacperturbations in a complex fashion. Neurons in intrathoracicextracardiac ganglia do not respond to cardiac perturbations in afashion similar to that displayed by intrinsic cardiac ones. Consistentcoherence of activity generated by differing populations of neurons hasbeen identified among medullary and spinal cord sympathetic efferentpreganglionic neurons, as well as among different populations ofsympathetic efferent preganglionic neurons. A relatively low level ofinputs on a spatial scale to one population of intrathoracic cardiacneurons results in low coherence among its components. In contrast,excessive input to this spatially distributed nervous systemdestabilizes it, leading for instance to cardiac arrhythmia formation.Since neurons in one part of the intrathoracic neuronal network respondsolely to inputs from a single region of the heart, such as frommechanosensory neurites in a right ventricular ventral papillary muscle,then the potential for imbalance within the different populations ofneurons in various levels of the intrathoracic neuronal hierarchyarises.

[0136] Ultimately, the outflows of efferent neuronal signals to thevarious regions of the heart depend to a large extent on the direct orindirect inputs they receive from cardiac and major intrathoracicvascular sensory neurites in addition to pulmonary mechanosensoryneurites. The redundancy of function and non-coupled behavior displayedby neurons in intrathoracic extracardiac and intrinsic cardiac gangliaminimizes the dependency for regional cardiac control on a singlepopulation of intrathoracic neurons. This may be particularly relevantwith, respect to supporting the output of the ischemic heart. In thatregard, network interactions occurring among intrathoracic extracardiacand intrinsic cardiac neurons secondary to inputs from cardiovascularafferent neurons involve local circuit neurons feeding informationforeword to cardiac parasympathetic and sympathetic efferent neurons.These network interactions are under the constant influence of spinalcord neurons

CARDIAC AFFERENT NEURONS

[0137] Overview of cardiac sensory neuronal transduction. It has beenknown for some time that cardiac sensory neurites (nerve endings) areassociated with somata located in ganglia relatively distant from theheart, nodose and dorsal root ganglia. It has recently become evidentthat cardiac sensory neurites are also associated with somata located inintrathoracic ganglia, including those on the heart. The relativedistance between these sensory neurites and their associated somatarepresents a major determinant of their function. The somata of manycardiac afferent neurons located near to or on the target organ displayhigh frequency (phasic) activity that directly affects target organefferent neurons (TABLE I). In this manner, high fidelity informationcontent can exert rapid control over efferent neurons adjacent to or onthe heart that modulate regional contractility. In contrast, cardiacafferent neuronal somata located relatively distant from their sensoryneurites (i.e., in nodose or dorsal root ganglia) are, of necessity,involved in longer latency influences on second order neurons in thecardiac neuroaxis. These relatively distant cardiac afferent neurons, assuch, are involved in relatively long latency cardio-cardiac reflexes,being spatially removed from the target organ they display memory. Adivision of cardiac sensory neuronal function into two broad, functionalcategories can be based on spatially derived cardiac sensorytransduction (TABLE I). It should be noted that some cardiac sensoryneurons within dorsal root ganglia generate high frequency phasicactivity, particularly when their sensory neurites are exposed toincreasing concentrations of local chemicals. TABLE I Cardiac afferentneuronal function Fast responding afferent Slow responding neuronsafferent neurons Mechanosensory specific Multimodal(mechanical/chemical) Activity related to local Not responsive tomechanical events instantaneous events High frequency, phasic Tonic, lowfrequency (non-tonic) activity activity High fidelity signals Noisysignals that limit resolution Noise free transduction Requires noise forsignal transduction Limited memory Memory capability (affected by pastevents) Soma located primarily on Soma primarily in or ganglia distantfrom the heart Near the heart Primarily inputs to short Primarily inputsto control longer control loops Loops

[0138] That two broad categories of cardiac afferent neurons exist(TABLE I) indicates unique transduction capabilities such that cardiacinformation provided to second order cardiac neuroaxis neurons dependsnot only on the location of their sensory neurites, but on the locationof their somata. The sensory information transduced by fast respondingcardiac afferent neurons, impinging as it does directly on cardiac motorneurons, is one of the primary determinants of the input function tocardiac efferent neurons that coordinate regional cardiac behavior. Fastresponding cardiac sensory neurons normally generate relatively highfrequency (10-100 Hz) activity patterns reflective of regionalcardiodynamics. Slow responding cardiac afferent neurons generallytransduce alterations primarily in the local chemical milieu and, thus,by their nature are generally not responsive to regional alterationsthat occur on a short time scale. During physiological states, theygenerate tonic activity at lower frequencies (0.1-1 Hz).

[0139] The sensory neurites associated with intrinsic afferent neuronalsomata are located in atrial and ventricular tissues, as well as theadventitia of major coronary arteries. The sensory neurites associatedwith the somata of afferent neurons in intrathoracic extracardiacganglia are concentrated in the same cardiac regions, in addition tobeing found around the origins of vena cava and on the thoracic aorta.Cardiovascular afferent neurons within the thorax provide feed-forwardinformation to efferent neurons in intrathoracic ganglia, some via localcircuit neurons. Those in the nodose ganglion influence medullarynucleus tractus solatarius neurons, while those in dorsal root gangliainfluence spinal cord neurons. As discussed hereinabove, the variedtransduction properties displayed by cardiac afferent neurons in nodose,dorsal root and intrathoracic ganglia reflect to a considerable extentthe anatomical location of their somata, i.e., the distance betweentheir somata and associated sensory neurites. Cardiac afferent neuronswith somata close to or on the heart influence cardiac efferent neuronsto initiate short-loop reflexes with short latencies of activation whilethose located in nodose ganglia initiate longer latency reflexes. Thatis why cardiac afferent neurons in intrathoracic ganglia displaydifferent transduction capabilities than those in dorsal root and nodoseganglia.

[0140] Afferent axons arising from cardiac or intrathoracic vascularsensory neurites vary in diameter (degree of myelination), according tothe location of the cardiopulmonary nerve in which they course. Forinstance, most aortic mechanosensory neurites are associated with Adaxons, most of which are located in the intrathoracic dorsalcardiopulmonary nerve. Many ventricular sensory neurites are associatedwith c class axons. On the other hand, carotid artery mechanosensoryneurites associated with afferent axons are divisible into the Ad and Cfiber categories, each population displaying unique transductionproperties.

[0141] Function. The majority of cardiac sensory neurons, particularlythose located distant from the heart, generate sporadic, low frequencyactivity. As the activity generated by the most cardiac afferent neuronsis of low frequency (i.e., 0.01-0.1 Hz), information content cannotreside in the interspike intervals of activity. Rather, it residesprimarily in their average activity over time unless their activitybecomes entrained to cardiodynamics in the presence of increased sensoryneurite chemical milieu. Information transduced by multimodal sensoryneurites associated with each axon connected to individual cardiacafferent neuronal somata also depends on their cardiac spatialdistribution. Arrays of atrial sensory neurites are concentrated in theregion of the sino-atrial node (right atrium) and the dorsal aspects ofboth atria, others are scattered throughout the rest of the atria.Ventricular sensory neurites are concentrated in the outflow tracts ofthe two ventricles as well as the right and left ventricular papillarymuscles. Another concentration of sensory neurites is located in theadventitia on the inner arch of the thoracic aorta.

[0142] Intrathoracic cardiac afferent neurons influence (viaintrathoracic local circuit neurons) cardiac efferent postganglionicneurons with latencies as short as 40 milliseconds. Nodose ganglioncardiac afferent neurons influence cardiac parasympathetic efferentpreganglionic neurons in the medulla via short latency reflexes (75 ms)as well. On the other hand, dorsal root ganglion cardiac afferentneurons influence sympathetic efferent postganglionic neurons via longerlatency (100-500 ms) reflexes. Thus, the differing populations ofcardiac sensory neurons located at each level of the cardiac neuroaxisnot only displaying unique transduction characteristics, but subservecardio-cardiac reflexes that of necessity differ in latency and form.

[0143] Nodose ganglion afferent neurons. Using neuroanatomical tracingtechniques, about 500 somata associated with cardiac sensory neuriteshave been identified throughout the right and left nodose ganglia. Theiraxons belong to the Ad and c classes, as defined by Erlanger and Gasser.Histochemical evidence indicates that the somata of nodose ganglionafferent neurons express receptors for a variety of neurochemicals,including adenosine, bradykinin and substance P receptors. Most of thesecardiac afferent neurons transduce multiple chemicals, includingpurinergic agents such as adenosine (FIG. 12). Few nodose ganglioncardiac sensory neurons solely transduce alterations in the mechanicalmilieu of the heart.

[0144] Doral root ganglion afferent neurons. Despite the widely heldopinion that the majority of cardiac afferent neurons are locatedprimarily in left-sided dorsal root ganglia, anatomic evidence indicatesthat cardiac afferent neurons are distributed relatively equally amongright and left dorsal root ganglia from the C6 to the T6 levels of thespinal cord. Afferent neuronal somata lie scattered predominantly, butnot exclusively, around the centrally located axons in these ganglia.Over 500 cardiac sensory neurons have been identified anatomically incanine dorsal root ganglia from the T₁ to the T₃ levels of the spinalcord, ganglia containing up to 50 cardiac afferent neuronal somata. Theaxons connecting cardiac sensory neurites with somata in dorsal rootganglia belong to the Ad or c classes of axons, each having littlebearing on their sensory transduction capabilities.

[0145] Intrathoracic extracardiac ganglion afferent neurons. Functionalevidence indicates the presence of cardiac sensory neuronal somata instellate, middle cervical and mediastinal ganglia. Axons connectingatrial or ventricular mechanosensory neurites with somata inextracardiac ganglia belong in the Ad class of axons. Those connectingintrathoracic vascular mechanosensory neuritis with somata inintrathoracic extracardiac ganglia belong to Ad class of axons as well.On the other hand, ventricular endocardial mechanosensory neuritesconnected with somata in intrathoracic ganglia belong to c class axons.Cardiac and aortic chemosensory neurites connected with somata inintrathoracic ganglia also belong to Ad class axons.

[0146] Intrinsic cardiac ganglion afferent neurons. Unipolar neurons arelocated throughout atrial and ventricular intrinsic cardiac ganglionatedplexuses. Based on anatomical and functional data, the somata of someintrinsic cardiac afferent neurons project axons centrally; theremainder interacting directly with other intrinsic cardiac neuronsexclusive of central neuronal inputs. Sensory neurites associated withintrinsic cardiac afferent neurons are located in all four chambers ofthe heart (particularly in the cranial aspect of the ventricles) aremultimodal in nature (transduce mechanical and chemical stimuli).Unfornunately, little is currently known of their transductioncapabilities.

[0147] Cardiac afferent neurons with sensory neurites located primarilyin the atria and the outflow tracts of the ventricles or majorintrathoracic vessels initiate short, intermediate and relatively longduration cardiovascular-cardiac reflexes, depending on their multimodaltransduction capabilities.

INTRATHORACIC EXTRACARDIAC AND INTRINSIC CARDIAC GANGLIONIC INTERACTIONS

[0148] Cardiac sensory input to the multiple nested feedback loopswithin the intrathoracic cardiac neuronal axis displaying redundancy offunction and non-coupled behavior within the different anatomical levelsof this hierarchy (FIG. 1) to minimize dependency of regional cardiaccontrol on a single population of neurons. The different populations ofcardiac afferent neurons, being capable of transducing multiple stimuli,forms the basis for integrated control of cardiac efferent neuronsaffecting regional cardiac function. Such control resides from the levelof target organ to that of the central nervous system. As mentionedhereinabove, neurons in intrathoracic extracardiac and intrinsic cardiacganglia exhibit differential reflex control over regional cardiacfunction that depends in large part on the varied anatomy and functionof afferent neurons providing information about the cardiac milieu. Thisconcept is based on the observation that intrathoracic extracardiac andintrinsic cardiac neurons display redundancy of function and non-coupledbehavior (FIG. 13), such non-coupled behavior minimizing cardiacdependency on a single population of intrathoracic neurons.Intrathoracic reflexes can exert considerable influence over regionalcardiodynamic behavior (11).

[0149] Intrathoracic cardiac afferent neurons are multimodal in nature(i.e., responsive to local mechanical and chemical stimuli), transducinga host of chemicals that include ion channel modifying agents (i.e.,veratridine; c.f., FIG. 13), β1- or β₂-adrenoceptor agonists, a ₁- or a₂-adrenoceptor agonists, excitatory amino acids, or peptides (c.f.,angiotensin II, bradykinin or substance P). The activity generated bypopulations of intrinsic cardiac local circuit neurons display, as aconsequence of such sensory inputs, periodically occurring coupledbehavior (cross correlation coefficients of activities that reach, onaverage, 0.88±0.03; range 0.71-1) for 15-30 seconds periods of time.This coupled activity occurs every 30-50 seconds during basal states, aswell as when cardiac afferent neuronal inputs to this neuronal hierarchyincrease in response to alterations in the ventricular chemical milieu.

[0150] On the other hand, neurons in intrathoracic extracardiac (middlecervical or stellate) and intrinsic cardiac ganglia do not display suchfunction, despite the fact that neurons in intrathoracic extracardiacand intrinsic cardiac ganglia receive inputs from cardiac mechanosensoryand chemosensory neurites. That is due in part because neurons inintrathoracic extracardiac ganglia receive many inputs mechanosensoryneurites located on the inner arch of the aorta. That some of theseneurons are still influenced by cardiac sensory inputs whendecentralized from central neurons indicates that intrathoracic cardiacafferent neurons can influence the intrathoracic neuronal hierarchyindependent of central neuronal inputs.

[0151] Neurons in intrathoracic extracardiac and intrinsic cardiacganglia exhibit non-coupled behavior, even when they are mutuallyentrained to cardiac events by cardiovascular afferent feedback (FIG.13). This shows a redundancy of cardioregulatory control exerted by thedifferent populations of intrathoracic neurons. That these differentpopulations respond differently to similar cardiac interventionsindicates the selective nature of the feedback mechanisms extant indifferent ‘levels’ of the intrathoracic neuronal hierarchy FIG. 1. Thisalso implies minimal reliance at any time on one population ofperipheral autonomic neurons for the control of regional cardiacbehavior. The selective influence exerted by each population ofintrathoracic (intrinsic and extrinsic) neurons on regional cardiacfunction depends in large part on the nature and content of their inputsfrom cardiac and intrathoracic vascular sensory neurites. Since thesensory information transduced by most cardiac sensory neurons is in the0.1 Hz range, it is unlikely that meaningful data is represented byinterspike intervals during physiological states as such relatively lowfrequency activity is not coherent. The fact that most of the sensoryinformation they receive is of low frequency content implies that theirresponsiveness is dependent primarily on average activity rather thaninstant-to-instant activity change (interspike intervals). Coherent(rhythmic) activity is generated by limited populations of cardiacsensory neurons such as those in dorsal root ganglia. Indeed, excessivesensory neuronal input to spinal cord neurons in the ischemic state mayact to destabilize cardiac neuronal hierarchical control of cardiacelectrical behavior.

INTRATHORACIC SYNAPSES

[0152] Direct application of neurochemical agonists or selectiveantagonists has been used to survey receptor subtypes associated withneurons within the intrathoracic cardiac nervous system and tocharacterize the functional differences of neurons within its variousganglia. Chemical stimulation of specific intrathoracic neurons with lowdoses of chemicals such as nicotine, neuropeptides, catecholamines,amino acids and purinergic agents can induce changes in their activity.When neuronal changes so induced are of sufficient magnitude,alterations in cardiac pacemaker, conductive and regional contractilefunction occur. The cardiac responses so induced reflect activation ofspecific populations of neurons in intrathoracic extracardiac orintrinsic cardiac ganglia as similar application of such neurochemicalsto intracardiac axons of passage does not effect neuronal activity orcardiodynamics. In agreement with that, transection of all extrinsicneuronal inputs to the intrathoracic nervous system (acutedecentralization) attenuates cardiac responses so elicited. This dataindicates the importance of the connectivity of neurons within thethorax with central ones in mediating cardiocardiac reflexes.

[0153] Cholinergic Mechanisms: Synaptic transmission in cardiacautonomic ganglia has been thought to be principally involved in therelease of acetyicholine by presynaptic terminals and subsequent bindingof that neurotransmitter to nicotinic cholinergic receptors onpostganglionic neurons. In mammals this synaptic junction is notobligatory, indicating that a significant convergence of inputs may benecessary to evoke postganglionic activity. Nicotinic and muscariniccholinergic agonists and antagonists modify intrinsic cardiac neurons invitro and in vivo, as well as neurons in intrathoracic extracardiacganglia. Local application of nicotine to intrinsic cardiac orintrathoracic extracardiac neurons induces alterations in cardiac rateand regional contractile function. Activation of intrinsic cardiacneurons with nicotine induces either augmenter or depressor cardiaceffects, depending on the population of neurons so affected. Blockade ofnicotinic receptors attenuates, but does not eliminate, these cardiacreflexes. Muscarinic cholinergic blockade attenuates synaptic functionwithin intrathoracic ganglia, as well, indicating that acetylcholineexerts both mediator and modulator effects at synaptic junctions withinintrathoracic ganglia.

[0154] Noncholinergic Mechanisms: Blockade of nicotinic cholinergicreceptors attenuates, but does not eliminate synaptic transmissionwithin intrathoracic ganglia indicating that non-nicotinic synapses actas primary mediators of synaptic transmission within the intrathoracicnervous system. Anatomical and physiological studies have identifiedmultiple putative neurotransmitters in association with neuronal somatain mammalian intrathoracic extracardiac and intrinsic cardiac ganglia.These chemicals include purinergic agents (adenosine and ATP), alpha-and beta-adrenergic agonists, angiotensin II, bradykinin, calcitoningene-related peptide, neuropeptide Y, histamine, serotonin, substance Pand vasoactive intestinal peptide as well as excitatory and inhibitoryamino acids. Many of these putative neurotransmitters arise from neuronswhose cell bodies are intrathoracic extracardiac (stellate and middlecervical) ganglia, while other may be synthesized by neurons intrinsicto the heart. Direct application of various putative neurotransmittersadjacent to neurons in intrinsic cardiac or intrathoracic extracardiacganglia modifies cardiac pacemaker and contractile activities. Suchresponses presumably reflect varied receptor mediated activation ofadjacent neurons and their associated dendrites since when identicalconcentrations of these neurochemicals are applied directly tointracardiac axons of passage neuronal activity and cardiac indicesremain unaffected.

[0155] Thus, when taken together, synapses interconnecting intrathoracicafferent, local circuit and efferent neurons utilize a host ofneurochemicals in the regulation of regional cardiodynamics, even whendisconnected from the influence of central neurons.

MEMORY FUNCTION WITHIN THE INTRATHROACIC NERVOUS SYSTEM

[0156] Cardiovascular-cardiac reflexes exert long-term control overcardiodynamics, including those initiated solely within theintrathoracic neuronal hierarchy. Intrathoracic cardio-cardiac reflexesdisplay different latencies of activation in as much as intrathoraciccardiac afferent neurons influence local circuit neurons in ganglia atdifferent levels of the thorax (intrinsic cardiac, mediastinal, middlecervical and stellate ganglia) following different latencies. Thediffering populations of cardiac afferent neurons that initiate thesevaried intrathoracic reflexes display unique transductioncharacteristics that are suited to the cardio-cardiac reflexes that theysub serve. For instance, intrathoracic cardio-cardiac reflexes havelatencies as short as 40 ms, whereas those involving spinal cord neuronshave latencies that exceed 450 millisecond. Thus, the relative distancebetween cardiac sensory neurites and neuronal somata is a significantdeterminant of cardio-cardiac reflex latencies they initiate.

[0157] Inherent in this issue is the fact that intrathoracic neuronsinvolved have two types of memory: (1) The first type involvesnon-computational memory displayed by cardiac chemosensory neurons, inas much as the previous status of their transduction behavior is a majordeterminant of their responsiveness to a chemical stimulus. The slowlyvarying, long-term transduction capabilities exhibited by cardiacchemosensory neurites is characteristic of passive memory. (2) Thesecond type of memory displayed in the intrathoracic nervous system isrepresented by active processing of sensory inputs from: (i)cardiovascular afferent neurons and (ii) central efferent preganglionicneurons. This computational memory resides in the network interactionsthat are dependent on intrathoracic local circuit neurons. Their statedependent memory represents hysteretic computation of cardiovascularsensory information that, along with inputs from central neurons, exertsongoing control over cardiac efferent neurons. Such computation abilityis necessary if a population of neurons is to simultaneously processinformation arising from many sources—an important characteristic of theintrathoracic nervous system. The presence of such complex informationprocessing within the intrathoracic autonomic nervous system has led tothe discovery that this nervous system functions as a distributiveprocessor of centripetal and centrifugal information arising from andgoing to the heart that, of necessity, requires state-dependent memory.

[0158] Memory displayed by slow responding cardiac afferent neurons.Most of the cardiac afferent neuronal somata located near or on thetarget organ transduce high frequency (phasic) information directly totarget organ efferent neurons that control regional contractilebehavior. In this manner, high fidelity information content can exertrapid control over cardiac efferent neurons coordinating regionalcontractile patterns. On the other hand, the cardiac sensory neuriteslocated anatomically distant from their associated somata (i.e., innodose and dorsal root ganglia) take longer to influence second orderneurons (c.f., neurons in the CNS). These latter cardiac afferentneurons are involved in relatively long latency cardio-cardiac reflexes.Presumably because of that function (lack of necessary short terminfluences), for the most part they display relatively long term memoryfunction since they transduce slowly varying chemical signals. It shouldbe noted that some intermediary cardiac afferent neurons also generatetonic activity, only generating high frequency, phasic activity whenexposed to increasing concentrations of chemicals reflective of theirmultimodal transduction properties. The passive memory functiondisplayed by these slow responding cardiac sensory neurons resides inthe state dependent properties of their cardiac chemosensory neurites.This is also indicative of the fact that chemical excitation of theseafferent neurons remains long after removal of the stimulus—yet anotherform of memory.

[0159] Local circuit neuronal memory function. It has been postulatedthat active memory resides in the multi-synaptic processing of cardiacsensory information that takes place within the intrathoracic neuronalhierarchy. This is particular relevant with respect to the processing ofcardiopulmonary sensory information by intrathoracic local circuitneurons. It has been determined that memory is displayed by remodeledintrathoracic local circuit neurons following chronic removal of theircentral neuronal inputs. Short duration (10 msec.) cardiopulmonarysensory inputs to neurons in chronically decentralized intrathoracicganglia results in the activation of local circuit neurons therein forup to 2 seconds. Such data indicates a memory capacity that lasts for anumber of cardiac cycles subsequent to sensory inputs arising during onecardiac cycle.

[0160] As there is little direct relationship between sensory inputs andoutput in the intrathoracic cardiac nervous system, its local circuitneurons act to compute state-dependent information on a beat-to-beatbasis. This permits inputs from multiple sources (peripheralcardiovascular afferent neurons and spinal cord efferent neurons) toinfluence restricted cardiac efferent neuronal outputs to the heart inan efficient manner and over time. Thus, one function of intrathoraciclocal circuit neurons is key to understanding hysteretic informationprocessing (memory) since their capacity to compute relatively minorsensory input alterations without adapting out represents an importantcharacteristic of this neuronal hierarchy. In such a scenario, localcircuit neurons function to reduce ‘noise’ to ensure restricted (notexcessive) output in the presence of multiple sensory inputs.

[0161] This processing of cardiovascular sensory information byintrathoracic local circuit neurons accounts for the stability of thecontrol exerted over regional cardiac function during relativelyprolonged period of time in normal cardiovascular states. On the onehand, simple state switching among excitatory versus inhibitory neuronsin this population would generate oscillatory behavior such as occursamong excitatory and inhibitory neurons in the spinal cord. This would,in fact, lead to instability of function since computational analysiswould become deranged and noise reduction capabilities would be lost. Onthe other, memory function associated with intrathoracic local circuitneurons, driven by ongoing cardiac sensory inputs, ensures stablecontrol over cardiac efferent neuronal outputs. For that reason,hysteretic memory related to the active processing of cardiac sensoryinformation is important for the ultimate stability of cardiac efferentneuronal control.

[0162] Current data indicates that passive memory resides in cardiacsensory transduction and active memory in the processing of thatinformation by local circuit neurons within the intrathoracic neuronalhierarchy. Control based memory residing in intrathoracic extracardiacand intrinsic cardiac local circuit neuronal interactions, driven asthey are by cardiovascular sensory inputs, it is not a passive process.Neurons in chronically decentralized intrathoracic ganglia also displayhysteretic memory. In the situation where there is a loss of inputs fromcentral neurons, 100 millisecond long bursts of sensory information(such as arise from aortic mechanosensory neurites during each cardiaccycle) affect local circuit neurons for up to 3 seconds after theirdiscontinuance. That period of time is sufficient for the next five orsix cardiac cycles to be generated.

[0163] Thus, events in one cardiac cycle influence regional cardiacbehavior throughout a few subsequent cardiac cycles via fed-forewordreflexes residing solely within the intrathoracic nervous system. Byutilizing such fed-foreward reflexes, the intrinsic cardiac nervoussystem can be pre-conditioned through the use of SCS or DCA to thereby“override” quench neuronal signals which would place the heart into adiseased state. Such pre-conditioning may take the form of constant SCSor DCA stimulation; and/or long pulses of SCS or DCA stimulationfollowed by short or long resting periods. In this manner the intrinsiccardiac nervous system is pre-conditioned to resist ischemic neuronaloverloading.

FOCAL VENTRICULAR ISCHEMIA

[0164] Myocardial ischemia. The importance of the processing of sensoryinformation arising from the ischemic myocardium by the intrathoraciccardiac nervous system in the maintenance of adequate cardiac output isonly now beginning to be appreciated by those of ordinary skill in theart. One of the major challenges to neurocardiology is understanding theresponse characteristics of each component of the cardiac neuronalhierarchy to myocardial ischemia so that focused neurocardiologicalstrategies can be devised to stabilize cardiac function in such a state.For that reason, one of the objects of the present invention is toremodel the heart using SCS or DCA stimulation to combat remodeling thatoccurs within the intrathoracic cardiac nervous system in the presenceof focal ventricular ischemia. The selective nature of the responseselicited by each component of the intrathoracic neuronal hierarchy tomyocardial ischemia depends on how each population is affected by thecontent of their altered cardiac sensory inputs. Neuronal interactionsin diseased states are relevant given the fact that pharmacologicalagents proven for use in treating heart failure (i.e., beta-adrenoceptoror angiotensin II receptor blocking agents) target not onlycardiomyocytes directly, but also indirectly by altering their inputsfrom cardiac efferent neurons secondary to altering the intrathoracicneuronal interactions.

[0165] Data also indicates that the cardiac neuronal hierarchy becomesobtunded by a variety of interventions, including multiple transmurallaser ‘revascularization’ therapy or heart failure. Intrathoracicneuronal function also remodels in the presence of focal ventricularischemia. Given the fact that certain populations of intrathoracicneurons, when activated, can induce ventricular fibrillation even in thenormally perfused heart, therapy directed at the intrinsic cardiacnervous system, whether pharmacological or surgical in nature, orthrough use of SCS or DCA stimulation are of benefit in managing theischemic heart and one of its sequellae—ventricular arrhythmias.

[0166] Activation of the dorsal columns of the cranial thoracic spinalcord results in a suppression of the activity generated by neurons notonly on the target organ, but also in middle cervical and stellateganglia. It is known that neurons in middle cervical and stellateganglia are under the constant influence of spinal cord neurons suchthat following their decentralization the activity generated by many ofthe latter increased upon removal of such control. Furthermore, removalof spinal cord inputs to the intrathoracic extracardiac nervous systemresults in enhancement of many intrathoracic extracardiac cardio-cardiacreflexes. It has also been shown that excessive activation of spinalcord neurons suppresses the intrinsic cardiac nervous system i.e.preconditioning the intrinsic cardiac nervous system.

[0167] Heart failure has been considered to be primarily a hemodynamicdisorder. Only recently has the importance of neurohumoral mechanismsthat act to maintain adequate cardiac output in the presence of heartfailure become appreciated, particularly with respect to arrhythmiaformation. This recognition and other clinically relevant findings haveforced a reappraisal of neuronal mechanisms involved in regulating theischemic myocardium.

[0168] Upper cervical neuronal modulation of upper thoracic cellactivity and interactions within and between upper cervical and upperthoracic spinal neurons involved in this processing have been examined.More specifically, these experiments have determined that differentpopulations of neurons within and between segments of the spinal cordexhibit coherence and correlation of activity and may, on occasion, actindependently. It has been determined that neurons in the upper cervical(C1-C2) spinal cord are organized to process cardiac sensory informationand coordinate the interactions between the C1-C2 and the T3-T4 spinalneurons, to thereby determine autonomic outflow to the intrinsic cardiacnervous system. Coupling of neuronal processing fluctuates within andbetween two cell populations during increased cardiac sensorystimulation. In the present application it is shown that chemicalactivation of upper cervical neurons modulates the stimulus locked andlong lasting responses of thoracic spinal cord neurons to myocardialalgogenic chemical stimuli. It has also been demonstrated that cardiacsensory information arising via thoracic sympathetic afferent activityascends in the spinal cord via propriospinal neurons to influenceneurons in the upper cervical spinal cord. In addition, vagal sensoryinputs excite neurons in upper cervical spinal segments. Thus, the uppercervical spinal cord is an area that processes cardiac sensoryinformation transduced by afferent somata in nodose and dorsal rootganglia. Based on these experiments and data, it was determined that,within the hierarchy of control that regulates cardiac function (FIG.1), neurons in C1-C2 spinal cord process cardiac sensory information tocoordinate the interactions within and between C1-C2 and T3-T4 spinalneurons and thereby determine autonomic outflow to the intrinsic cardiacnervous system.

[0169] Well-established evidence links the autonomic nervous system tolife-threatening arrhythmias and cardiovascular mortality. The autonomicimbalance of increased sympathetic activity and reduced vagal activityincreases the likelihood for ventricular fibrillation during myocardialischemia. Clinical studies using various markers of impaired vagalactivity supports the experimental evidence that this type of autonomicimbalance increases cardiovascular risk. Disease processes may changethe balance between the central and peripheral neurons involved in suchregulation. For instance, when the activity generated by cardiac sensoryneurons becomes abnormal, cardiac function can be affected profoundly.Therefore, a disturbance of the fine balance within the cardiac neuraxiswill produce dramatic changes in cardiac efferent neuronal outflow.Within the hierarchy of central neurons that control the heart, complexsensory processing involves spatial and temporal summation of cardiacsensory inputs to affect central preganglionic autonomic efferentneurons that modulate autonomic efferent postganglionic activity, aswell as intrathoracic ganglionic reflexes and the intrinsic cardiacnervous system. Experimental studies, as discussed herein, show thatpathological processes can change the integrative behavior of thecentral cardiac neuraxis. For example, arrhythmias generated byocclusion of the coronary artery are significantly decreased aftertransection of upper thoracic dorsal roots. This observation indicatesthat the spinal cord receives and processes information that isgenerated during an ischemic episode. Furthermore, spinal neurons of theupper thoracic segments are sensitive to changes associated witharrhythmias. These changes can occur when cardiac sensory neurites areactivated intensely and for long periods when cardiac tissue becomesdamaged during regional ventricular ischemia.

INFORMATION PROCESSING OF SPINAL NEURONS—SINGLE CELL ANALYSIS

[0170] Sympathetic afferents from the heart convey noxious andmechanical, presumably innocuous, information via the dorsal rootsprimarily in the upper thoracic segments. We herein show that bothcentrally projecting as well as non-projecting neurons respond tonoxious stimuli applied to the heart. We also demonstrate that chemicalstimulation of cardiac nociceptors produces either a stimulus-locked orlong lasting evoked response of superficial and deep spinal neurons ofthe upper thoracic spinal cord.

[0171] The classical concept of acute cardiac nociception is based on aserial neuronal system that transmits information from cardiac afferentsto spinal neurons. The transfer of information is mediated by classicalneurotransmitters, such as excitatory amino acids, that lead to membranepotential changes within a time span of milliseconds to seconds. Thus,nociceptive stimulation of cardiac afferents evokes discharge rates ofspinal neurons that increase as long as the nociceptors are stimulated.It is generally assumed that impulses of spinal neurons responding tocardiac stimuli constitute a simple renewal process with a very highnumber of degrees of freedom. Electrophysiological studies show thatnociceptive responses of spinal neurons are the basis of mean dischargerates of single neurons. It was further shown that discharge rates areoften correlated in a generally linear manner to the intensity ofnoxious stimulation and antinociception is consequently defined as areduction in discharge rates of nociceptive neurons.

MULTIPLE CELL ANALYSIS

[0172] Under normal, physiological conditions stimuli applied to theheart do not elicit marked changes in cardiac efferent neuronal activitybecause central neurons suppress excessive cardiac sensory informationprocessing. In the hierarchy of cardiac control, activation of spinalneuronal circuits modulate the intrathoracic cardiac nervous system.Activation of the dorsal columns at the T1-T2 segments significantlyreduces the activity generated by the intrinsic cardiac neurons in theirbasal conditions as well as when activated in the presence of focalventricular ischemia induced by occluding the left coronary artery. Notonly does dorsal column activation modulate the intrinsic cardiacnervous system, but it also modifies the activity of spinal neuronswithin the T3-T4 segments. In addition, the central nervous systemmaintains a tonic inhibitory influence over intrathoraciccardiopulmonary-cardiac reflexes. Reflexes mediated through the middlecervical ganglion are increased after decentralization. Thus, diseaseprocesses change the balance between the central and peripheral neuronalprocessing of cardiac sensory information. For instance, when theactivity generated by cardiac sensory neurons becomes excessive, e.g.during focal ventricular ischemia, cardiac function can be profoundlyaffected. Thus, a disturbance of the fine balance within the cardiacneuraxis results in dramatic changes in cardiac efferent neuronalactivity. Nests of neural networks in the hierarchy of cardiac control,therefore, appear to interact effectively when an appropriate balance isachieved therein.

[0173] Upper Cervical Modulation of the Thoracic Spinal Cord and Heart

[0174] Within the hierarchy for cardiac control, neurons of the uppercervical segments modulate information processing in the spinal neuronsof the upper thoracic segments. In human studies, spinal cordstimulation of the C1-C2 spinal segments relieves pain symptoms inpatients with chronic refractory angina pectoris. Experimental studiesdisclosed and discussed herein show that spinal cord activation of theupper cervical segments of the spinal cord suppresses the activity ofspinal neurons in T3-T4 segments. Furthermore, chemical stimulation withglutamate of cells in the C1-C2 segments also reduces upper thoracicspinal neuronal activity and that chemical stimulation of C1-C2 cellssuppresses the activity of lumbosacral spinal neurons. It is especiallyimportant to note that this suppression of lumbosacral neuronal activityis sustained even after the spinal cord is transected at thespinomedullary junction. Glutamate was chosen as the stimulant becauseit only activates cell bodies but not the axons passing through theupper cervical segments.

[0175] Neuroanatomy of high cervical neurons

[0176] Little information about descending pathways that originate fromC1-C2 segments is available, but anatomical studies provide someevidence for a subpopulation of C1-C2 cells that are involved inpropriospinal modulation of spinal sensory neurons. Horseradishperoxidase (HRP) injection into the thalamus of cats labeled cells inthe lateral cervical nucleus; however, a subpopulation of cells in themedial part of this nucleus was unlabeled, and axons of these unlabeledcells appear to descend to caudal spinal segments. Others in the arthave confirmed those descending projections by injecting HRP in theC8-T5 segments of one monkey and finding labeled cells in the lateralcervical nucleus and in the C1-C2 gray matter. Furthermore, anothergroup skilled in the art have shown in cats that neurons in the medialportion of the lateral cervical nucleus respond to noxious stimuli. Inaddition, spinal sensory neurons in upper cervical segments receivenoxious inputs from large areas of the body and thus, may project tomore caudal spinal segments, as well as to the thalamus. Indeed, it hasbeen proposed that the lateral spinal nucleus, which extends down theentire spinal cord, may participate in inhibition of efferent nerveactivity in rats. The data as presently disclosed also shows that afterselective spinal transections in rats supported the concept that spinalinhibitory effects in sensory neurons utilize upper cervical segments.

[0177] Spinal Relay for Vagal Inputs

[0178] A very interesting finding is the differential processing ofcardiac vagal afferent information in the cervical and thoracic spinalcord. Electrical and chemical stimulation of vagal afferent fibersprimarily excites neurons of the C1, C2 segments. It should be pointedout that these cervical cells also receive input that was carried to thethoracic spinal cord via the sympathetic afferents. However, the vagalinformation elicits larger evoked responses. In contrast to excitationof upper cervical spinal neurons, vagal input from the cardiopulmonaryregion generally reduces neuronal activity in sensory cells of rats,cats and monkeys in segments below C3; vagal facilitation of responsesto noxious inputs are reported only at low stimulus intensities.Antinociceptive effects of vagal stimulation also are found in thetail-flick response in rats, and vagotomy attenuates opioid-mediated andstress-induced analgesia.

[0179] Disruption of the C1-C2 neurons with the excitotoxin, ibotenicacid, eliminates the suppressor effects on thoracic spinal neurons withvagal stimulation. Vagal suppression of evoked activity of thoracicspinal neurons resulting from intrapericardial injections of algogenicchemicals is attenuated or eliminated after ibotenic acid was placed onthe dorsal surface of the C1-C2 spinal segments.

[0180] Neurons in the upper cervical (C1-C2) and upper thoracic (T3-T4)spinal cord process cardiac sensory information to coordinate theinteractions within and between these populations of spinal cord neuronsand thereby modulate efferent neurons that regulate regional cardiacfunction. Specifically, neurons in C1-C2 spinal cord process cardiacsensory information to coordinate the interactions within and betweenC1-C2 and T3-T4 spinal neurons and thereby determine autonomic outflowto the intrinsic cardiac nervous system.

[0181] Simultaneous Recordings of Two Neurons

[0182] Different populations of neurons within and between segments ofthe spinal cord exhibit coherence and correlation of activity and may,on occasion, act as independent units. Valuable data was gathered byrecording from two cells simultaneously by using two microelectrodes.FIG. 14 demonstrates simultaneous recordings of two cells andparticularly indicates the correlation of two cells in the T3 segment ofthe spinal cord.

[0183] Noxious Chemical Stimulation of the Heart-Responses of T3-T4 andC1-C2 Neurons

[0184] A typical example of an upper thoracic cell responding to somaticand noxious chemical stimulation of the heart is shown in FIG. 15. Thechemical evoked responses also show tonic activity descending from uppercervical and supraspinal regions. The evidence of tonic modulationsupports the conclusion that there is a hierarchy of control ormodulation from the upper cervical spinal cord (FIG. 1).Intrapericardial injections of algogenic chemicals generally increasethe activity of T3-T4 spinal neurons, but the activity appears todecrease in a few cells. Intrapericardial injections also increased theaverage activity of 50% of the C₁-C₂ spinal neurons from 8.1±1.3 imp/sto 21.6±2.6 imp/s. Mechanical stimulation of the somatic fields on thechest and forelimbs activated afferent fibers that converged onto theT3-T4 spinal neurons; whereas, the input from somatic afferent fibersconverging onto C1-C2 neurons was from receptive fields in the neck andjaw regions.

[0185] Cell Response to Coronary Artery Occlusion

[0186] Chemical stimulation of the heart using algogenic chemicalstimulation of cardiac afferents provides a global method for activatingcardiac afferents. The effects of coronary artery occlusion on uppercervical and upper thoracic cell activity is included herein because itspecifically provides a means of activating nociceptive afferents inregional areas of the heart and clearly demonstrates that SCS or DCAstimulation has a preconditioning or protective effect on the heartthereby dampening neuronal activity of the intrinsic cardiac nervoussystem. Such an effect leads or lends itself to therapeutic SCS or DCAstimulation of the intrinsic cardiac nervous system to prevent and/orlessen the effects of cardiac pathologies. FIG. 16 demonstrates that theleft coronary artery can be occluded and thereby produce a response in aT3 spinal neuron.

[0187] Neurochemistry

[0188] Experiments were performed to show changes in c-fos expression inthe upper thoracic segments in response to activation of cardiacafferents by injecting algogenic chemicals into the pericardial sac(FIG. 17). In the resting conditions, very little c-fos was expressed inthe T3-T4 segments and the little c-fos that was expressed appeared inthe more superficial laminae (I-III) rather than in the deeper laminae,where cells are activated by stimulation of cardiac afferent fibers. Inanother experiment, an intra pericardial infusion of normal saline didnot cause any additional expression of c-fos. These results show thatvery few neuronal sites are activated by either the surgical proceduresor the infusion of a solution which does not activate the cardiacafferent fibers. Heart rate and mean blood pressure did not changeduring these infusions. In contrast, the intrapericardial infusion ofalgogenic chemicals produced greater c-fos expression in the superciallaminae and laminae surrounding the central canal V-VII (FIG. 17). Thisdata simulates a process (angina and the activation of cardiacnociceptive sympathetic afferent fibers) that most likely occurs for anextended time period. This data is provided to demonstrate that thesetechniques represent a reproducible approach to simulate cardiacpathologies and that the use of SCS or DCA stimulation to modulate theintrinsic cardiac nervous system can significantly impact theprogression or effecti of such cardiac pathologies.

[0189] Effects of Vagal Stimulation on c-fos Expression in C1-C2 Neurons

[0190] In order to determine whether input from the vagus would activateC1-C2 neurons, c-fos immunohistochemical studies following vagalelectrical stimulation were performed. Three groups have been evaluated:unoperated controls, rats with the vagus nerve crushed for 2 hrs, andrats with the vagus nerve stimulated with the following parameters: (20Hz, 30 V, 0.2 ms, 5 min on, 5 min off for 1 hr. Abundant c-fosimmunoreactive neurons were found in the superficial dorsal horn(marginal zone, substantia gelatinosa), nucleus proprius, central grayregion (area X), and ventral horn (FIG. 18).

[0191] C1 Modulation of Upper Thoracic Cell Activity

[0192] Originally, it was assumed that supraspinal pathways arenecessary for descending inhibitory effects of visceral afferents onsensory neurons. However, evidence shows that in rats that high cervicalneurons can mediate inhibitory effects of cardiopulmonary spinal inputin lumbar spinothalamic tract (STT) and dorsal horn (DH) neurons. Thus,it appears that the upper cervical segments play an important role inthe hierarchy that controls the efferent outflow to the intrathoracicand intrinsic cardiac nervous system. Based on this knowledge andevidence from previous studies, we conclude that cell bodies located inthe gray matter of C1-C2 spinal segments can modulate nociceptivecardiac-evoked activity of spinal neurons in the upper thoracic spinalcord. The effects of glutamate activation of cell bodies in the uppercervical spinal cord on the activity of cells in the T3-T4 spinal cordevoked by injections of bradykinin (BK) into the pericardial sac havebeen examined. Glutamate has been used to activate cell bodies in thecervical spinal cord by others in the art. Glutamate (1M) was absorbedonto filter paper pledgets (2×2 mm) and was placed on the dorsal surfaceof the C1-C2 segments. Saline control pledgets were applied at the samesites before and after glutamate. Saline did not elicit any responses.

[0193] Chemical Stimulation of C1-C2 Cells Before and After Rostral C1Transection

[0194] The evoked activity of one T3 cell to glutamate is shown before(FIGS. 19A-C) and after rostral C1 (FIGS. 19D-F) transections. Thesetransections demonstrate that supraspinal pathways are not necessary toelicit the effects from C1 cell activation. Chemical stimulation of theC1 cell bodies with glutamate suppresses the evoked responses of the T3cell to algogenic chemical stimulation of cardiac afferent fibers. Cellsin the upper cervical segments serve as an important relay in thehierarchy of cardiac control that modulates the activity of cells inthoracic segments.

[0195] Neuroanatomy and Immunohistochemistry

[0196] Retrograde tracing was used to detect upper cervical neurons withpropriospinal projections to the lumbosacral spinal segments in the rat.Termination sites of the upper cervical propriospinal neurons in thegray matter of the upper thoracic segments are shown in FIG. 20.Termination sites of the thoracic propriospinal neurons in the uppercervical segments are also shown in FIG. 21. Using PHA-L an anterogradestudy has been performed from the C1-C2 segments in the rat. PHA-L wasinjected into the C1-C2 segments and rats survived 12-24 hours.Anterogradely labeled fibers were identified in the nucleus proprius asfar as the upper thoracic segments. PHAL moves by fast transport,degrades rapidly, and is picked up by fibers of passage. The resultsshown in FIG. 20 indicate the feasibility of anterograde tracing fromupper cervical segments.

[0197] Vagal Modulation of T3-T4 Neurons via the C1-C2 Segments

[0198] Electrical Stimulation of the Vagus

[0199] Electrical stimulation of the vagal afferents, in general,suppresses the activity of the upper thoracic spinal neurons (FIG. 21).Electrical and chemical stimulation of vagal afferents excites uppercervical spinal neurons.

[0200] Chemical Disruption of the C1-C2 Neurons

[0201] Chemical disruption of the C1-C2 spinal neurons alters theeffects of stimulation of the cardiac afferent input on the regulationof information processing in the cervical and thoracic spinal cord. Inorder to produce chemical disruption of cells, ibotenic acid was chosenbecause it is an excitotoxin that has been used effectively in previousstudies. Ibotenic acid is a structurally rigid glutamate analog thatdestroys neuronal perikarya, but spares axons and non-neuronal cells.After ibotenic acid is injected into a nucleus or applied to the surfaceof the spinal cord, the cells in the region are initially excited andthen enter a phase of depolarization block. FIG. 22 shows that ibotenicacid applied to the dorsal surface of the C1-C2 spinal segments causesenergy impairment and/or apoptosis of cells located beneath the surfaceof these segments. The advantage of this methodology is that theneuronal relays in the C1-C2 segments can be disrupted withoutinterrupting the axons that pass through this region.

[0202] Vagal Effects After Chemical Disruption of C1-C2 Cell Bodies

[0203] At least part of the vagal inhibitory effects of the upperthoracic neurons depend on the C1-C2 relay. Approximately 20 min. afteribotenic acid was placed on the spinal cord, the inhibitory effects tovagal stimulation observed in FIG. 22 were eliminated. These resultsindicate that at least part of the vagal inhibitory pathway is dependenton an intact relay in the C1-C2 segments.

[0204] Using high frequency, low intensity electrical stimulation of thedorsal aspect of the T1-T2 spinal cord, the modulatory effects on thefinal common integrator of cardiac function, the intrinsic cardiacnervous system, have been determined. Dorsal cord activation by itselfdecreases basal intrinsic cardiac neuronal activity by 77%. Thissuppression of neuronal activity persisted for 30-45 minutes afterterminating the dorsal cord stimulation. When LAD occlusion wasinitiated during dorsal cord activation, neuronal activity remainedsuppressed. Thus, use of SCS or DCA cord stimulation to preconditionand/or remodel the neuronal activity of the intrinsic cardiac nervoussystem has been shown.

[0205] Thus, dorsal cord activation suppresses intrinsic cardiacneuronal activity in both normally perfused and ischemic hearts anddorsal cord activation suppresses the activity of upper thoracicspinothalamic tract neurons evoked by chemical stimulation of cardiacafferents. Dorsal cord activation or SCS can modulate the activity ofcells in central nervous system and the intrinsic cardiac nervoussystem. Dorsal cord activation can be used at either the thoracic or thecervical levels. The cervical segments are particularly interesting,because this is a key region for hierarchical control, and dorsal cordactivation of the upper cervical segments has been used to relieve thesymptoms in patients with chronic refractory angina pectoris. Dorsalcord activation of the upper cervical segments suppresses the responsesof a T3 spinal neuron evoked by algogenic chemical stimulation of thecardiac afferents is shown in FIG. 23.

[0206] Chemical stimulation of the upper cervical cell bodies suppressesupper thoracic cell responses to nociceptive (chemical) andnon-nociceptive (mechanical) input. In contrast, chemical stimulation ofthe upper thoracic cell bodies excites the upper cervical spinalneurons. Furthermore, the responses to nociceptive and non-nociceptivestimuli are enhanced. Inactivation of the upper cervical cell bodieseliminates the suppression of spontaneous and evoked activity of theupper thoracic neurons. In fact, the nociceptive and non-nociceptiveresponses are facilitated because elimination of the upper cervicalspinal neurons reduces the tonic inhibition that continually impinges onthe upper thoracic spinal neurons. Elimination of the upper thoraciccell bodies does not have an appreciable effect on the spontaneousactivity and the evoked responses of the upper cervical spinal neurons,because vagal input produces larger responses of the upper cervicalneurons than do the inputs that originate from sympathetic afferents.

[0207] Vagotomy also changes the modulation of spontaneous activity andnociceptor evoked responses of C1-C2 and T3-T4 spinal neurons.Inactivation of C1-C2 cell bodies eliminates vagal effects of chemicaland mechanical stimulation on the activity of the upper thoracicneurons. Vagotomy also eliminates the nociceptive and non-nociceptiveresponses of the C1-C2 spinal neurons after elimination of input fromthe T3-T4 spinal neurons. C-fos expression at the upper thoracicsegments increases after C1-C2 ablation before and after activation ofthe vagal afferents, because the cells of the upper cervical segmentstonically suppress cell activity in the upper thoracic spinal cord.Perturbations change the correlation characteristics of the pairs ofneurons. In addition, the responses of the individual neurons, which arerecorded simultaneously, change their response characteristicsindependently after the interventions are made. The cfos expression doesnot change in the cervical segments because of the disruption of thecells by ibotenic acid that participate in producing the suppression ofthoracic activity. In anterograde tracing studies with PHAL, a clearerpicture of the reciprocal innervation between the C1-C2 and T3-T4segments is seen.

[0208] The experiments were designed in order to study the activity andresponses of individual cells (192) as well as pairs (96) of cells.Results indicate that coronary artery occlusion evokes responses in theC1-C2 and T3-T4 neurons. That ischemic responses differ from thealgogenic chemical responses because chemical stimulation provides aglobal activation of the afferents; whereas, coronary artery occlusionlimits the stimulus to a specific region of the heart. Since vagal inputprovided the strongest input to the C1-C2 neurons and sympatheticafferents provided the excitatory inputs to T3-T4 neurons, differentpatterns of activity are demonstrated. Since activation of the vagusexcites C1-C2 cells and suppresses the activity of T3-T4 cells, vagotomyreduced the responses of upper cervical neurons but enhances upperthoracic responses to coronary artery occlusion. Coronary arteryocclusions will increase the number of cells filled with c-fosexpression in both C1-C2 and T3-T4 spinal segments.

[0209] The experiments were also designed to study the activity andresponses of individual cells (384) as well as pairs (192) of cells toaddress information processing of the effects of algogenic chemicalstimulation and coronary artery occlusion before and after the cells ofC1-C2 are disrupted using ibotenic acid. Dorsal cord activation of theT1-T2 or C1- C2 segments suppresses the evoked T3-T4 cell activity toalgogenic chemical stimulation and coronary artery occlusion. Sincedisruption of cells with ibotenic acid reduces or eliminates vagalsuppression of the evoked activity of the T3-T4 cells, inhibitoryeffects of dorsal cord activation are reduced or eliminated, becausesynaptic activity occurs in the same segments that are stimulatedelectrically with dorsal cord activation. Disruption of C1-C2 cells withibotenic acid might reduce the effectiveness of T1-T2 dorsal cordactivation on the evoked responses of T3-T4 spinal neurons due to thevasodilator effects of dorsal cord activation being eliminated when thespinal cord was transected at least four to six segments rostral to thesite of stimulation. Dorsal cord activation changes the correlation ofcell activity in the pairs of cells. These changes are responsible forthe suppressed activity of the intrinsic cardiac nerve activity. Dorsalcord activation generates patterns of activity in the spinal neuronsthat act to stabilize the activity generated by the intrinsic cardiacneurons.

[0210] Algogenic chemical stimulation evokes short lasting and longlasting excitatory as well as inhibitory responses of the C1-C2 andT3-T4 neurons. If two neurons recorded simultaneously receive commoninput from algogenic chemical stimulation of cardiac afferents, theyhave more synchronous action potentials than statistically expected, andtheir cross-correlation function correspondingly shows a sharp centralpeak (i.e. when the mutual delay is at zero). However, the central peakis widened to a variable extent when several neuronal connections areinterposed between the locus of common input and the neurons from whichthe activity is recorded. There are stronger correlations in pairs ofneurons when one neuron is in the superficial dorsal horn and the otherone is in the deeper dorsal horn. Experiments have shown that latency tothe onset of the evoked response of superficial cell to algogenicchemical stimulation of cardiac afferents is shorter than the latency tothe onset of the evoked response in a deeper cell. This difference inthe latency suggests that the superficial neurons serve as aninterneuron between the input from the primary afferents and theactivation of the deeper cells. Since vagal input provided the strongestinput to the C1-C2 neurons and the sympathetic afferents provided theexcitatory inputs to the T3-T4 neurons, different patterns of activityhave been demonstrated. Since activation of the vagus excites C1-C2cells and suppresses the activity of T3-T4 cells, vagotomy will reducethe responses of the upper cervical but enhance upper thoracic responsesto nociceptive algogenic chemical stimulation. No effects ocurred usingsaline controls. With respect to mechanical studies, some of the cellsdischarge in response to the premature ventricular contraction. Some ofthe bursts occur early in the compensatory phase, but more commonly theburst is associated with the potentiated contraction. Cells wereanalyzed individually and as pairs. Vagotomy does not prevent responsesof the neurons to chemical stimulation, but most likely modulates someof the mechanical responses. Chemical stimulation increases the numberof cells filled with c-fos expression in both the upper cervical andupper thoracic spinal segments. After bilateral vagotomy, a decreasednumber of cells with cfos, but the number of thoracic spinal cells withc-fos increases because vagal activation of upper cervical neuronssuppresses the activity in thoracic neurons. As shown in FIG. 18, cellslocated in specific regions of these segments were found.

[0211] Chemical stimulation of the upper cervical cell bodies suppressupper thoracic cell responses to nociceptive (chemical) andnon-nociceptive (mechanical) input. In contrast, chemical stimulation ofthe upper thoracic cell bodies excite the upper cervical spinal neurons.Furthermore the responses to nociceptive and non-nociceptive stimuli areenhanced. Inactivation of the upper cervical cell bodies eliminates thesuppression of spontaneous and evoked activity of the upper thoracicneurons. In fact, the nociceptive and non-nociceptive responses arefacilitated because elimination of the upper cervical spinal neuronsreduces the tonic inhibition that continually impinges on the upperthoracic spinal neurons. Elimination of the upper thoracic cell bodiesdoes not have much effect on the spontaneous activity and the evokedresponses of the upper cervical spinal neurons, because vagal inputproduces larger responses of the upper cervical neurons than do theinputs that originate from sympathetic afferents. Vagotomy changes themodulation of spontaneous activity and nociceptor evoked responses ofC1-C2 and T3-T4 spinal neurons. Inactivation of C1-C2 cell bodieseliminates vagal effects of chemical and mechanical stimulation on theactivity of the upper thoracic neurons. Vagotomy also eliminates thenociceptive and non-nociceptive responses of the C1-C2 spinal neuronsafter elimination of input from the T3-T4 spinal neurons. C-fosexpression at the upper thoracic segments increases after C1-C2 ablationbefore and after activation of the vagal afferents, because the cells ofthe upper cervical segments tonically suppress cell activity in theupper thoracic spinal cord. The perturbations change the correlationcharacteristics of the pairs of neurons. In addition, the responses ofthe individual neurons, but recorded simultaneously, change theirresponse characteristics independently after the interventions are made.The cfos expression is not changed in the cervical segments because ofthe disruption of the cells by ibotenic acid that participate inproducing the suppression of thoracic activity. Anterograde tracingstudies with PHAL, have shown that a clearer picture of the reciprocalinnervation between the C1-C2 and T3-T4 segments is obtained.

[0212] Differential remodeling of the peripheral and central cardiacnervous hierarchy and its nerve-cardiac myocyte junction in the presenceof a healed myocardial infarction, specifically as related to thegenesis of ventricular fibrillation occurs. Tests utilize a well-definedcanine model of ventricular fibrillation that combines three elementsrelevant to the genesis of malignant arrhythmias in man: a healedmyocardial infarction, acute myocardial ischemia, and physiologicallyelevated sympathetic efferent neuronal activity have shown thatdifferential remodiling is at least partially responsible for cardiacpathologies. Test also reveal and demonstrate that SCS or DCAstimulation of the intrinsic cardiac nervous system has apreconditioning effect pre-remodeling and a quenching effect postre-modeling. Based on an “exercise and ischemia test”, animals in thismodel separate into two groups: 1) animals that develop ventricularfibrillation and are thereby classified “susceptible” to fibrillation;and 2) dogs that don't develop sustained ventriculartachycardia/fibrillation and are thus defined as “resistant”. Thus,differential remodeling of the cardiac neuron hierarchy (central andperipheral) for reflex control of the heart occurs in susceptible versusresistant animals.

AUTONOMIC NERVOUS SYSTEM AND SUDDEN DEATH AFTER MYOCARDIAL INFARCTION

[0213] A canine model of lethal ventricular arrhythmias developed in1978 has been used to elaborate the mechanisms of sudden death aftermyocardial infarction (MI). In this model, animals with a chronicanterior wall infarction undergo a sub-maximal exercise stress test,culminating in transient total occlusion of the circumflex coronaryartery for 2 minutes. During that 2-minute period of transientmyocardial ischemia, 40% of the dogs develop ventricular fibrillation(VF); the remaining animals do not generate sustained ventriculararrhythmias. This model produces clinically relevant information byincorporating a healed anterior MI in the setting of elevatedsympathetic efferent neuronal tone (induced by exercise), coupled withacute, regional myocardial ischemia distant from the originalinfarction. This model was developed to duplicate the clinical situationof a patient with multi-vessel coronary artery disease who beginssub-maximal exertion in the convalescent phase of an uncomplicated MI,patients who then develop transient myocardial ischemia. In the dogmodel, those destined to develop VF display persistent tachycardia inresponse to transient, acute myocardial ischemia. In contrast, VFresistant animals have been found to possess active vagal reflexes thatcontrol heart rate during the ischemic event. Thus, this model producestwo distinct groups of animals, based on the occurrence of VF, that havevery different characteristics of autonomic control of heart rate.

[0214] This model gives rise to the data that non-invasive markers ofcardiac vagal reflexes predict risk for sudden death after myocardialinfarction. This is shown through baroreflex sensitivity (BRS) datarelating a rise in systolic blood pressure to RR interval slowing wasprospectively tested prior to exercise and the induction of myocardialischemia to predict outcomes. BRS was reduced in chronic MI dogsdestined to develop VF during exercise and acute, regional myocardialischemia. Interestingly, BRS was lower before MI in dogs that eitherdied after coronary artery ligation or developed VF within 30 days ofacute MI during exercise. Clinical conformation of these results hasbeen published and shows that BRS was lower in patients who subsequentlydied suddenly after their first myocardial infarction. Results establishthat autonomic markers add critical predictive information to the suddendeath risk profile after MI. Baroreflex sensitivity measurements provideone index of the cardiac parasympathetic nervous system. Heart ratevariability (HRV) quantifies cardiac autonomic interactions by measuringthe impact of vagally mediated respiratory sinus arrhythmia viabeat-to-beat RR interval variability derived from resting ECGrecordings. It is shown herein dogs at high risk for sudden death hadlow variability measurements, suggesting low tonic vagal input to theheart. Tonic autonomic activity was influenced significantly by MI andrecovered only in dogs at low risk for sudden death. In contrast, dogsat high risk for sudden death displayed little recovery during the first30 days following MI. This persistent blunting of vagal tone wasassociated with a high risk for VF during exercise and myocardialischemia. These experiments provide evidence that autonomic control ofheart rate remodels during the progression of coronary artery disease.

[0215] If depression of vagal efferent neuronal tone to the heart and,as a consequence, cardiovascular reflexes are important for thedevelopment of lethal ventricular arrhythmias, does augmentation ofcardiac vagal efferent neuronal activity prevent sudden cardiac death insuch a model? This issue was addressed using the Schwartz and Stonemodel of sudden death by electrically stimulating the vagus nerve bymeans of chronically implanted electrodes. When the vagosympathetictrunk was electrically stimulated during exercise initiated at the onsetof coronary artery occlusion, the incidence of VF was prevented in over80% of high-risk dogs tested. This effect was largely independent of theheart rate reduction associated with vagal activation. Furthermore,augmentation of tonic vagal activity by daily exercise trainingprevented VF in 100% of the high-risk dogs, either in the presence orabsence of acute myocardial infarction. Finally, left stellateganglionectomy was effective in reducing VF in these high-risk animals.Thus, abnormal autonomic control of the infarcted heart associated withsympathetic efferent neuronal dominance and weak vagal input, results inventricular electrically instability that increases the risk for suddencardiac death.

REMODELING OF THE CARDIAC NEURONAL HIERARCHY AFTER MYOCARDIAL INFARCTION

[0216] What comprises the cardiac neuronal hierarchy and why is itimportant for the management of cardiac arrhythmias in chronicallyinfracted hearts? Neurons in intrathoracic extracardiac and intrinsiccardiac ganglia have long been thought to act as simple efferentinformation relay stations involving one synapse, for instance inparavertebral sympathetic ganglia or parasympathetic ganglia on theheart. Recently, this concept has been extended in recognition of thefact that cardiovascular afferent information is also processed withinthe intrathoracic nervous system, including its component intrinsic tothe heart. Neurons in intrathoracic ganglia, including those on theheart, receive constant inputs from spinal cord neurons to modulatetheir behavior. They also receive sensory inputs from cardiac afferentneurons on an ongoing basis. That is why the activity generated by mostintrinsic cardiac neurons increases markedly in the presence increasedsensory inputs arising from the ischemic myocardium. Indeed, excessiveactivation of limited populations of intrinsic cardiac neurons inducedcardiac dysrhythmias that lead to ventricular fibrillation. Thus,therapies that act to stabilize heterogeneous evoked activities withincardiac reflex control circuits such, as the SCS or DCA stimulation ofthe intrinsic cardiac nervous system of the presently claimed anddisclosed invention, has obvious clinical importance.

[0217] Proper information exchange among the intrathoracic components ofthe cardiac nervous system act in concert to stabilize the electricaland mechanical behavior of the heart, particularly in the presence offocal ventricular ischemia. Different populations of neurons,distributed spatially within the intrathoracic cardiac nervous system,respond to cardiac perturbations in a coordinate fashion. If neurons inone part of this neuronal axis respond to inputs from a single region ofthe heart, such as the mechanosensory neurites associated with a rightventricular ventral papillary muscle, then the potential for imbalancewithin the different populations of neurons regulating various cardiacregions occurs and, thus, its neurons display little coherence ofactivity. On the other hand, relatively low levels of specific inputs ona spatial scale to the intrathoracic cardiac nervous system results inlow basal coherence among its various neuronal components, therebyacting to stabilize cardiac regulation. Alternatively, excessive inputto the spatially distributed intrathoracic nervous system destabilizescardiac electrical behavior, leading to cardiac arrhythmia formation.Intrathoracic extracardiac and intrinsic cardiac neurons receive tonicinputs not only from cardiac and major intrathoracic vascular sensoryneurites, but also from spinal cord neurons in the integration ofefferent neuronal inputs to the heart.

CHRONIC VENTRICULAR ISCHEMIA AND THE CARDIAC NEURONAL HIERARCHY

[0218] The infarct matrix is important in determining risk for VF in theSchwartz and Stone model discussed hereinabove. This is illustrated bydata showing epicardial conduction mapping across the infarct zone inhigh and low risk dogs. It has been found that conduction delays aremuch more profound across the infarct zone in high-risk dogs (>85millisecond) compared with low risk animals (FIG. 24). High-risk dogsexhibit “mottled” myocardial infarcts that are electrophysiologicallyunstable, with electrical activation waves persisting as long as 85milliseconds after epicardial electrical activation terminates. Thismatrix reflects a very large surface area for the development andsustaining of reentrant arrhythmias, which lead to VF in high-risk dogs.When ventricular function is normal, very fast VT leading to VF arisesfrom a purely reentrant mechanism. Components that contribute todevelopment of a mottled infarct include: autonomic characteristics ofdogs before MI. Baroreflex sensitivity is lower in dogs destined to dieafter MI or develop VF during the exercise and myocardial ischemia test.Using a marker derived of both baroreflex sensitivity and spectralanalysis of heart rate variability, dogs at high risk for post-MI suddendeath were identified with high sensitivity and specificity. Thesefindings indicate that innate differences in cardiac autonomic controlthat can be identified before the development of overt cardiac diseasemay determine post-MI sudden death. Furthermore, autonomic differencesbefore MI influence the type of infarct that develops with the LADligation in this model. This underscores the importance of understandingthe hierarchy of autonomic control of the heart and how abnormalitiescontribute to the pathophysiology of cardiac disease (FIG. 1).

[0219] The importance of the peripheral cardiac nervous system in themaintenance of normal cardiac output can be appreciated from thepresently claimed and disclosed invention. The selective nature of theresponses elicited by each component of the intrathoracic neuronalhierarchy to myocardial ischemia depends on how each population ofperipheral autonomic neurons is affected, as well as the nature andcontent of their sensory inputs. That ischemia sensitive cardiacafferent neurons in nodose and dorsal root ganglia influence thebehavior of central autonomic neurons which, in turn, modifycardiovascular autonomic efferent preganglionic neurons represents yetanother level of this regulatory hierarchy.

[0220] Myocardial ischemia. Recent anatomical and functional dataindicate the presence of the multiple neuronal subtypes withinintrathoracic extracardiac and intrinsic cardiac ganglia. Its intrinsiccardiac component functions as a distributive processor at the level ofthe target organ. The redundancy of function and non-coupled behaviordisplayed by neurons within intrathoracic extracardiac and intrinsiccardiac ganglia minimizes the dependency for such control on a singlepopulation of peripheral autonomic neurons. In that regard, networkinteractions occurring at the level of the heart integrateparasympathetic and sympathetic efferent inputs with local afferentfeedback to modify cardiac rate and regional contractile forcethroughout each cardiac cycle. A recent editorial by David Lathrop andPete Spooner of the NIH highlights the potential clinical relevance ofaltered processing of information by these populations of neurons suchthat a lack of coordination of data exchange within the cardiac neuronalaxis may lead to the genesis of cardiac arrhythmias.

[0221] Interactions Among Neurons in the Cardiac Neuronal Hierarchy

[0222] The different populations of neurons distributed spatially withinthe intrathoracic cardiac nervous system respond to cardiacperturbations in a complex fashion. For instance, neurons inintrathoracic extracardiac ganglia do not respond to cardiacperturbations in a similar fashion as intrinsic cardiac ones. Consistentcoherence of activity generated by differing populations of neurons hasbeen identified among medullary and spinal cord sympathetic efferentpreganglionic neurons, as well as among different populations ofsympathetic efferent preganglionic neurons. If neurons in one part ofthe intrathoracic neuronal network respond solely to inputs from asingle region of the heart, then the potential for imbalance within thedifferent populations of neurons in various levels of the intrathoracicneuronal hierarchy might occur. A relatively low level of inputs on aspatial scale to populations of intrathoracic cardiac neurons wouldresult in a low basal coherence among its components and stabilize thatsystem. In contrast, excessive input to this spatially distributednervous system would destabilize it, leading for instance to cardiacarrhythmia formation.

[0223] Arterial reflexes can become blunted during the evolution ofheart disease. Focal ventricular ischemia is known to altercardio-cardiac reflexes. Furthermore, ischemia induced liberation ofchemicals such as adenosine or hydroxyl radicals within the affectedmyocardium can suppress ventricular myocyte electrical and contractilebehavior. On the other hand, locally released adenosine or hydroxylradicals can influence the cardiac nervous system via excitation of itsafferent neuronal components. Thus, when devising a therapy to modifythe outcome of myocardial ischemia one must consider not only alteredcardiac myocyte behavior, but autonomic neuronal alterations. A briefsummary of some of the issues concerning autonomic neuronal control ofthe ischemic myocardium is presented below, including its importance inone sequellae of myocardial ischemia—ventricular arrhythmia formation.

[0224] Symptomatology. The somata of isolated afferent neurons aresensitive to adenosine. ATP and, to a lesser extent, adenosine influencesensory neurites of dorsal root ganglion neurons. The importance ofadenosine in the genesis of cardiac pain became evident when ChristerSylvén and his colleagues administered adenosine into the blood streamof patients with diseased coronary arteries. Indeed, the symptomsinduced by adenosine in these patients mimicked those that theyexperienced during effort. These data are in accord with the fact thatdorsal root ganglion purine cardiac afferent neurons play an importantrole in the genesis of pain and that the ventricular sensory neurites ofthese neurons become non-responsive to ischemia in the presence ofadenosine receptor blockade.

[0225] Cardiovascular reflexes secondary to myocardial ischemia.Alterations in heart rate secondary to ventricular ischemia can be due,in part, to altered neural control of cardiac pacemaker cells.Myocardial ischemia can be attended by not only by tachycardia, but alsoby bradycardia.

[0226] Most ventricular sensory neurites associated with nodose ganglioncardiac afferent neurons are sensitive to purinergic agents. Activationof a sufficient population of nodose ganglion afferent neurons byexposing their sensory neurites to purinergic agents can result in theinduction of bradycardia via medullary reflexes. Bradycardia can also beinduced when sufficient populations of intrinsic cardiac neuronsprojecting axons to medullary neurons are activated by purinergicagents. In contrast, activation of cardiac sensory neurites associatedwith dorsal root ganglion neurons with adenosine results in the reflexexcitation of sympathetic efferent neurons that innervate the heart. Thedetails of the various reflex responses induced when specificpopulations of cardiac afferent neurons in nodose as opposed to dorsalroot ganglia are modified by local ischemia remain to be fullyelucidated. Coordination of autonomic outflows to the heart depends to alarge extent upon the sharing of inputs from higher centers concomitantwith interactions among neurons in various intrathoracic ganglia. Thatsharing of cardiac afferent information occurs within the intrathoracicand brainstem/spinal cord feedback loops of FIG. 1 allows for overallcoordination of cardiac function.

[0227] Cardiac arrhythmias. Another sequel of myocardial ischemia is thedevelopment of cardiac arrhythmias. As neurons from the level of theinsular cortex to the intrinsic cardiac nervous system can be involvedin the genesis of cardiac arrhythmias, it is important to recognize thatsuch neurons can induce untoward cardiac electrical events in thepresence of myocardial ischemia. For instance, activation of arelatively minor population of intrinsic cardiac neurons in anesthetizedcanine preparations by exogenous application of an alpha- orbeta-adrenoceptor agonist, endothelin I or angiotensin II can induceventricular dysrhythmias or even fibrillation. DCA and SCS does reduceor ameliorate these effects.

NEURAL SUBSTRATES FOR ARRHYTMIA FORMATION IN ISCHEMIA

[0228] The selective nature of the responses elicited by each componentof the cardiac neuronal hierarchy to focal, ventricular ischemia dependson how each population of neurons within this autonomic neuronalhierarchy is affected and that depends in large part on the nature andcontent of their ventricular sensory inputs. It also depends, in part,on any alteration in ventricular efferent postganglionic axon functionsecondary to their presence within the ischemic zone.

[0229] Cardiac Afferent Neurons

[0230] The chemical milieu of the sensory neurites associated withintrinsic cardiac afferent neurons also change when the blood flow in acoronary artery is compromised. Locally liberated adenosine, ATP, oxygenfree radicals and peptides can affect the sensory neurites associatedwith afferent neuronal somata in nodose, dorsal root or intrathoracicganglia. Oxygen free radicals also affect the functional integrity ofventricular nerves. The quantities of purinergic agents liberated intothe local blood stream and pericardial fluid, increases duringventricular ischemia, as peptides or hydrogen peroxide can affect theactivity generated by intrathoracic and central cardiac afferent neuronsin an indirect fashion as chemicals accumulated in myocardial tissuesand pericardial fluid modify their sensory neurites. When coronaryarterial blood flow is restored, during the reperfusion phase variousmetabolites that accumulate upstream can influence intrinsic cardiacneurons and their sensory neurites supplied by that blood even more.

[0231] That ischemia sensitive cardiac afferent neurons in relativelydistant (nodose and dorsal root) ganglia versus the somata of cardiacafferent neurons relatively closer to the affected tissue (intrathoracicextracardiac and intrinsic cardiac afferent neurons) influence thebehavior of cardiac efferent postganglionic neurons via central andintrathoracic local circuit neurons represents yet another issue ofimportance within this regulatory hierarchy (FIG. 1). Alterations inheart rate secondary to ventricular ischemia activation of cardiacafferent neurons results in altered neural control of cardiac pacemakercells. Thus, myocardial ischemia can be attended by tachycardia orbradycardia. Activation of a sufficient population of nodose ganglionafferent neurons by exposing their sensory neurites to a variety ofchemicals that are liberated by the ischemic myocardium results in theinduction of bradycardia via medullary reflexes. In contrast, excitationof the cardiac sensory neurites associated with dorsal root ganglionneurons by chemicals such as adenosine results induces the reflexexcitation of sympathetic efferent neurons that innervate the heart.

[0232] Intrinsic cardiac neurons. Intrinsic cardiac neurons are modifiedby myocardial ischemia in two fashions: one direct and the otherindirect. Transient occlusion of the coronary arterial blood supply to apopulation of intrinsic cardiac neurons directly affects the function oftheir somata and/or dendrites. Presumably a lack of energy substratesnormally available to them via their local arterial blood supplyaccounts in part for their altered behavior, as well as the fact thatthey are bathed by local products of ischemia such as oxygen freeradicals and purinergic agents. Each major intrinsic cardiacganglionated plexus on human or dog hearts is perfused by two or morearterial branches arising from different major coronary arteries.Intrinsic cardiac neurons and cardiomyocytes are affected by hypoxia.Myocardial ischemia of short duration affects not only cardiac myocytefunction, but also the capacity of intrinsic cardiac neurons to respondto their sensory inputs. Metabolites accumulating locally when theregional coronary arterial blood supply of intrinsic cardiac neurons iscompromised also influence the somata and dendrites of such neurons in adirect manner. Thus, regional ventricular ischemia influences thecardiac neuronal hierarchy in a number of ways, depending on whether thearterial blood supply affected by the arterial lesion directly affectsthe somata and dendrites of somata therein or indirectly via affectingsensory neurites in the infarct zone.

[0233] Data indicate that adaptations occur within the cardiac neuronalhierarchy in the presence of acute, focal ventricular ischemia. Thecardiac nervous system remodels during chronic ischemic/infarction tomaintain control over regional cardiac dynamics.

[0234] Myocardial infarction is induced by ligation of the left anteriordescending coronary artery in an open chest procedure during surgicalanesthesia. The circumflex coronary artery is instrumented with apneumatic occluder so that reversible myocardial ischemia can be inducedat a later time. After 30 days of recovery, dogs have autonomic testsperformed including baroreflex sensitivity (Sleight phenylephrinemethod) and heart rate variability (time and frequency domain). Thenanimals run on a treadmill using a protocol in which workload (beltspeed and elevation) are increased every 3 minutes. Once heart ratereaches 210 beats per minute the circumflex occluder is inflated for 2minutes, the first minute the dogs continue to run on the treadmill andthe treadmill is stopped for the last minute. Forty percent of thepost-MI animals develop ventricular fibrillation (VF) during the 2minutes of coronary occlusion. The other 60% do not have sustainedventricular arrhythmias. An example of the arrhythmia that susceptibledogs develop is illustrated in FIG. 25. This observation indicates thatreflex vagal activation is relatively weak in susceptible dogs and thusleads to ventricular electrical instability and even ventricularfibrillation. This indication was further tested by measuring baroreflexsensitivity during activation of cardiac vagal fibers by means of highpressure baroreflex testing. Relating the heart rate slowing in responseto systemic hypertension (phenylephrine induced) quantifies baroreflexsensitivity (FIG. 26). It was found that baroreflex sensitivity wasdepressed in susceptible dogs compared with resistant animals and thebaroreflex was an accurate predictor of the outcome of the exercise andischemia test (Table II). TABLE II Resistant Susceptible Strong vagalreflexes Weak vagal reflexes High baroreflex sensitivity Low baroreflexsensitivity High heart rate variability Low heart rate variabilityTransmural scar Mottled scar No late potentials + late potentials

[0235] These findings were clinically validated in the multicenter trialcalled ATRIAMI in which baroreflex sensitivity was found to be anindependent risk factor for post-MI sudden cardiac death. Subsequently,the indication that weak vagal reflexes was responsible for susceptibledogs developing VF was tested using electrical stimuli delivered tovagal efferent preganglionic axons to augment cardiac vagal control.Vagal stimulation was started at the time of coronary artery occlusionand continued until the occluder was released. Vagal stimulationprevented VF in over 80% of the susceptible dogs. Even during subsequentexercise testing in which vagal stimulation was coupled with atrialpacing to maintain heart rate at control levels, VF was prevented inabout 50% of the animals. Therefore, electrical stimulation of cardiacvagal efferent neurons prevents ventricular electrical instability thatdevelops during exercise and transient myocardial ischemia insusceptible dogs.

[0236] One susceptible and one resistant dog were implanted with aspinal cord stimulator and allowed to recover for 7 days. Controlexercise and ischemia testing and heart rate variability were studiedprior to and during dorsal cord activation (DCA, 50 Hz, 200 ps, 90%motor threshold). The stimulator was activated for 4 hours daily for 4days; then testing was repeated with the stimulator on. FIG. 27 showsthe chronotropic response to graded increases in treadmill exercise.Once heart rate reaches 210 beats per minute the circumflex occluder isinflated for 2 minutes, the first minute the dogs continue to run on thetreadmill and the treadmill is stopped for the last minute. Whileconcurrent DCA minimally affected heart rate responses in the resistantdog (right panel), in the susceptible dog DCA reduced the heart rateduring the ischemic period (left panel).

SPINAL CORD INFLUENCES ON NEURAL CONTROL OF CHRONOTROPIC FUNCTION

[0237] Both spectral analysis (FIG. 28) and time domain analysis (FIG.29) of heart rate variability indicate that spinal cord stimulation viaDCA augments parasympathetic nervous system activity to the heart.

[0238] It is very difficult to predict how central and intrathoracicautonomic neurons involved in cardiac regulation remodel to sustaincardiac output in the presence of chronic, regional ventricularinfarction. Data indicate, however, that the cardiac neuronal hierarchybecomes obtunded by a variety of interventions, including chronicregional ventricular injury.

[0239] Information Processing within the Intrinsic Cardiac NervousSystem and its Control of Regional Cardiac Function

[0240] Myocardial ischemia and infarction induce substantial changes inthe intrathoracic nerve networks and their reflex control of regionalcardiac function. Chronic myocardial infarction/ischemia induces aheterogeneous distribution of efferent projections to cardiacend-effectors. Myocardial infarction/ischemia alters the neurochemicalprofile of that innervation, with differential increases in neuropeptidecontent within subsets of neurons contained within the intrinsic cardiacnervous system. The evolution of cardiac pathology is associated withdisruptions of the intrinsic cardiac nervous system and its ability toprocess afferent information and such changes will be more evident inthe CMVPG than the RAGP intrinsic cardiac ganglia. Animals that exhibitindices of higher vagal tone (higher baroreflex sensitivity and higherheart rate variability) demonstrate lesser degrees ofischemic/infarct-induced neural remodeling.

[0241] The Functional Connectivity of Intrinsic Cardiac andIntrathoracic Extracardiac Neurons in Normal and Acutely Ischemic Hearts

[0242] Little direct functional interconnectivity exists among intrinsiccardiac neurons and their intrathoracic extracardiac counterparts.Independent function as such indicates that little reliance on one suchpopulation normally occurs when regulating regional cardiac function;i.e., dysfunction of one population occurs without a major loss ofregional cardiac control. Significant alterations in the cardiac milieu,such as occurs during acute, focal ventricular ischemia, induces greatercoherence of activity among populations of intrathoracic andintrathoracic extracardiac neurons.

[0243] Chronic myocardial ischemia induces a heterogeneous distributionof efferent projections to cardiac end-effectors. We anticipate thatthis heterogeneous distribution of sympathetic fibers to the leftventricle results in similar heterogeneous release of catecholamines andneuropeptides into the interstitial space during stimulation of theefferent nerves. Finally, animals that exhibit indices of higher vagaltone (higher baroreflex sensitivity and higher heart rate variability)demonstrate lesser degrees of ischemic/infarct-induced remodeling of theefferent outflow of the left ventricle.

[0244] Activation of the dorsal columns of the cranial thoracic spinalcord suppresses the activity generated by neurons not only on the targetorgan, but also in middle cervical and stellate ganglia. It is knownthat neurons in these ganglia are under the constant influence of spinalcord neurons such that following their decentralization their activityincreases (i.e., spinal cord neurons exert tonic suppression of theirfunction), Removal of spinal cord inputs to the intrathoracic nervoussystem enhances many intrathoracic cardio-cardiac reflexes is tied tothe principle and thus excessive activation of spinal cord neuronssuppress the intrinsic cardiac nervous system.

[0245] Heterogeneous alterations within the intrinsic cardiac ganglia orat the end-terminus of the autonomic innervation to the ischemicmyocardium are major contributors to the increased incidence of suddencardiac death in patients with coronary artery disease. The increasedincidence of sudden death often result from lack of protection of themyocytes and instability of the cardiac electrical system. Chronic DCAameliorate ischemia-induced remodeling within the intrinsic cardiacnervous and thereby reduces the heterogeneous neural substrate thatpredisposes the susceptible animals to ventricular arrhythmias andsudden cardiac death.

[0246] Heart failure has traditionally been considered to be primarily ahemodynamic disorder. The importance of neurohumoral mechanisms that actto maintain adequate cardiac output in the presence of ventricularischemia is apparent. This recognition has forced a reappraisal ofneuronal mechanisms involved in regulating the ischemic myocardiumleading to the development of the presently claimed and disclosedinvention.

[0247] Spinal cord-peripheral neural interactions and modulation ofperipheral nerve function in the ischemic heart. Dorsal columnactivation stabilizes the intrinsic cardiac nervous system in acutemyocardial ischemia experiments were conducted. The purpose of theseexperiments was to determine if dorsal column activation (DCA) induceslong-term effects on the intrinsic nervous system, the final commonintegrator of cardiac function, particularly in the presence ofmyocardial ischemia. Methods: Activity generated by right atrial neuronswas recorded in 10 anesthetized dogs during basal states, and during 15min occlusions of the LAD coronary artery, with and without backgroundDCA. For DCA, dorsal T1-T4 spinal segments were stimulated for 17 min.at 90% of motor threshold (50 Hz; 0.2 ms duration). For combinedeffects, the coronary occlusion commenced 1 min into DCA. Results:Ischemia-induced excitatory effects on the intrinsic cardiac nervoussystem were suppressed (−76%) during DCA and for approximately 20 minafter DCA termination. Conclusions: DCA suppresses basal activity withinthe intrinsic cardiac nervous system and prevents the ischemia-inducedactivation of these peripheral neural networks. This stabilization ofintrinsic cardiac neuronal function, induced by higher elements of theneural hierarchy for cardiac control, is maintained for prolongedperiods post-stimulation and are reflective of the neural memory ofthese processes. These long-term effects may partially explain theprolonged effects patients with angina experience not only during DCA,but also for a time thereafter.

[0248] Coronary artery occlusion induces differential catecholaminerelease in the normal and ischemic myocardium. (FIG. 30, solid line).The purpose of this study was to determine if transient coronaryocclusion differentially effects norepinephrine (NE) and epinephrine(EPI) release into the canine ventricular interstitial space (ISF).Methods: In anesthetized dogs, left ventricular ISF samples werecollected by microdialysis during 15 min occlusions of the circumflexcoronary artery. Results: Coronary artery occlusion (CAO) induced abiphasic response in ISF catecholamine release, with ISF EPI increased400% and ISF NE increased 150% in both the normal and ischemicmyocardium. By 15 min of CAO, ISF catecholamines returned towardsbaseline. ISF EPI, and to a lesser extent NE, increased uponreperfusion. Conclusions: Coronary artery occlusion evokes adifferential release of catecholamines, primarily reflected in theneuronal release of epinephrine. Neuronal release of catecholamines intothe ISF, associated with coronary artery occlusion onset andreperfusion, is reflective of reflex interactions among peripheral andcentral components of the cardiac neural hierarchy in response to theischemic stress.

[0249] Doral column activation stabilizes peripheral adrenergic functionin acute myocardial ischemia. The purpose of this study was to determinewhether DCA modulates NE and EPI release into the canine ISF in bothnormal hearts and those exposed to transient myocardial ischemia.Methods: In anesthetized dogs, left ventricular ISF samples werecollected by microdialysis during electrical stimulation (50 Hz, 0.2 ms)of the dorsal T1-T4 segments of the spinal cord at an intensity of 90%of motor threshold with and without concurrent 15 min occlusions of thecircumflex coronary artery. Results: ISF EPI doubled by 10 min andtripled by 20 min of DCA (239 to 935 pg/ml, respectively). ISF EPIremained twice baseline 20 min post-DCA. DCA increased left ventricularNE by 43% (890 to 1273 pg/ml); ISF NE returned to baseline values 20 minpost-DCA. Heart rate and left ventricular inotropic function were notaffected by DCA. When 15 min CAO was instituted during DCA (FIG. 30,dotted lines), ischemia induced changes in ISF EPI and NE were obtunded(FIG. 30, solid lines), both at the onset of occlusion and duringreperfusion. Conclusions: DCA evokes differential release ofcatecholamines, primarily reflecting neuronal release of epinephrine.Evoked release of catecholamines into the ventricular interstitiumpersists for a considerable period of time post-DCA. Pre-existing DCAsuppresses the release of catecholamines by intrathoracic adrenergicneurons reflexly-induced by transient myocardial ischemia. The long-termDCA effects on myocardial catecholamine release may account, in part,for the fact that this form of therapy produces clinical benefit topatients with angina pectoris not only during its application, but for atime thereafter.

Spinal Cord-Peripheral Neuronal Interactions Modify MyocardialElectrical Stability. Dorsal Column Activation Reduces VentricularFibrillation Accompanying Acute Myocardial Ischemia.

[0250] The purpose of this study was to determine if the stabilizationof peripheral neural function exerted by DCA reduces the potential forventricular fibrillation induction in acute myocardial ischemia.Methods: Under anesthesia, atrial and ventricular electrograms wererecorded during basal states, and during 15 min occlusions of theproximal circumflex artery with and without pre-existing DCA. DCAinvolved electrical stimulation (50 Hz, 0.2 ms) of the dorsal T1-T4segments of the spinal cord at an intensity of 90% of motor thresholdfor 36 min, with coronary artery occlusion commencing 15 min into DCA(FIG. 31). Results: Coronary artery occlusion induced ventricularfibrillation (VF) in 5 of 10 dogs, with VF occurring within 6 min ofreperfusion (FIG. 31 arrows). With pre-existing DCA, coronary arteryocclusion induced VF in 1 of 9 dogs, the VF occurring after terminatingDCA. Conclusions: DCA stabilizes efferent neuronal outflows for cardiaccontrol and obtunds the ischemia-induced reflex activation of cardiacneural networks. Such stabilization of neural function reduces thesubstrate for induction of lethal arrhythmias during acute myocardialischemia and, in particular, the subsequent reperfusion period.

[0251] Dorsal column activation stabilizes ischemic myocardialelectrical dysfunction. The purpose of this study was to determinewhether DCA modulates electrical imbalance within the chronicallyischemic ventricle. Methods: An ameroid constrictor was implanted aroundthe left circumflex coronary artery to gradually occlude that vessel.Four weeks later, under general anesthesia multiple ventricular unipolarelectrograms were recorded in the normal and ischemic left ventricleduring basal states and when ANG II (40 μg/min; 1 minute) wasadministered to right atrial neurons before and after DCA. Results: ANGII increased the area and magnitude of regional ST segment changes inthe ischemic ventricle. ANG II induced minimal changes in the electricalbehavior of the normal myocardium. DCA (50 Hz, 0.2 ms, 0.32 mA for 15min) modified ischemic indices, even suppressing regional ventricular STsegment abnormalities previously induced by ANG II. Conclusions: DCAsuppresses ischemia induced ventricular electrical disturbances. Thismay occur, in part, via stabilizing intrathoracic adrenergic neuronsthat modulate the ischemic ventricle.

[0252] Processing of cardiac sensory information by neurons in the upperthoracic (T3-T4) spinal cord. Chemical activation of cardiac receptorsdifferentially affect activity of superficial and deeper spinal neuronsin rats. The purpose of this study was to evaluate responses ofsuperficial (depth<300 μm) versus deeper thoracic spinal neurons tochemical stimulation of cardiac afferent neurons and to determine ifdescending central neuronal inputs modulate these effects. Methods:Extracellular potentials of single T3-T4 neurons were recorded inpentobarbital anesthetized, paralyzed and ventilated male rats. Acatheter was placed in the pericardial sac to administer 0.2 ml of analgogenic chemical mixture that contained adenosine (10⁻³ M),bradykinin, histamine, serotonin, and prostaglandin E₂ (10⁻⁵ M).Results: Intrapericardial chemicals elicited responses in 27% of thesuperficial neurons and in 47% of the deeper neurons. All superficialneurons that responded to cardiac afferents were excited. Of the deeperneurons, approximately 80% were excited, 15% were inhibited and 5%showed excitation-inhibition. Spontaneous activity of superficialneurons with short-lasting excitatory responses was significantly lowerthan that of deeper neurons (P<0.05). After cervical spinal transection,spontaneous activity generated by superficial and deeper neuronsincreased significantly, as did responses to chemical activation ofcardiac afferents neurons. Conclusions: Chemical stimulation of cardiacafferent neurons excites superficial T3-T4 spinal neurons; deeperneurons exhibit multiple patterns of responses. These data furtherindicate that thoracic spinal neurons that process cardiac nociceptiveinformation are tonically inhibited by higher center neurons.

[0253] Descending modulation of thoracic cardiac nociceptivetransmission by upper cervical spinal neurons. The purpose of this studywas to examine effects of stimulating upper cervical spinal neurons onspontaneous and evoked activity of thoracic spinal sensory neurons thatresponded to noxious cardiac stimuli. Methods: Extracellular potentialsof single T3 neurons were recorded in pentobarbital anesthetized malerats. A catheter was placed in the pericardial sac to administerbradykinin (10⁻⁵ M, 0.2 ml, 1 min) as a noxious cardiac stimulus andsaline as control. A glutamate pledget (1 M, 1-3 min) was placed on thesurface of C1-C2 segments to chemically activate upper cervical spinalneurons. Results: In 77% of the T3 neurons tested, glutamate at C1-C2inhibited spontaneous activity and/or excitatory responses tointrapericardial bradykinin. After transection at the rostral C1 spinalcord, excitatory amino acid (glutamate) excitation of C1-C2 neuronsstill reduced the spontaneous activity of T3 neurons, as well asexcitatory inputs from cardiac sensory neurons. Conclusions: Chemicalactivation of C1-C2 spinal neurons evokes a descending inhibition inthoracic spinal cord cardiac neurons during basal states as well as inthe presence of noxious cardiac stimuli. Furthermore, modulation ofcranial thoracic neurons by upper cervical spinal neurons does notrequire supraspinal connectivity.

[0254] Interdependence of cardiac sensory information processing byneurons in the upper thoracic (T3-T4) spinal cord. The purpose of thesestudies was to evaluate the coordination of activity among upperthoracic neurons that process cardiac sensory inputs. To date, we haveevaluated the correlation of spontaneous and evoked activity of 15 pairsof T3 spinal neurons. Included is FIG. 32 that demonstrates the abilityto simultaneously record the activity generated by two cells in the T3segment of the spinal cord, both before and after their multisynapticvagal inputs were disrupted. In this case, vagotomy changed correlationamong their function. In another pair of T3 neurons, their activityrecorded simultaneously demonstrated that they exhibited coordination ofactivity approximately 130 ms from time 0, a feature that disappearedafter placing glutamate on the C1-C2 segments of the dorsal spinal cord(not shown). In that case, background activity was similar before andafter glutamate application. This data indicate that coordination ofactivity among T3 neurons can be modified by cardiac afferent inputs orfollowing activation of descending pathways. These novel data indicatethat different populations of neurons distributed spatially within andamong thoracic and upper cervical spinal cord segments respond tocardiac sensory inputs in a coordinate fashion. These results alsodemonstrate the contributions that vagal afferent neurons make to thecoordination of activity among cervical and thoracic spinal neurons;their cardiac component representing an important transducer ofmyocardial ischemic events to central neurons.

[0255] Mechanical activation of spinal neurons using programmedventricular arrhythmias. Data demonstrating the feasibility of recordingthe responses of spinal neurons to premature ventricular contractionsand compensatory beats. To generate these events, an electrical stimuluswas applied through a pair of stainless steel electrodes that wereinserted in the free wall of the left ventricle. FIG. 33 shows that theT3 deeper spinal neuron responded with a burst of activity during thecompensatory beat. However, the cell was unresponsive to mechanicalevents associated with normal beats. The results demonstrate that we areable to record the activity of cells in response to the effects ofadministering an extra stimulus electrically.

[0256] Without the present specification, one of ordinary skill in theart would not have appreciated or known to use SCS or DCA stimulation asa means to (1) electrically influence the intrinsic cardiac nervoussystem to protect cardiac myocytes from initial ischemic damage or frombeing further damaged during subsequent ischemic episodes; and (2)preserve the electrical stability of the intrinsic cardiac nervoussystem and the heart itself prior, during, or post an ischemic episode.As such, the presently claimed and disclosed invention would benon-obvious in light of the prior art showing the use of SCS stimulationfor the treatment of angina. In fact, it is widely and traditionallybelieve by those of ordinary skill in the art that SCS alleviated anginapain by either changing blood flow within ischemic or non-ischemicmyocardium or modifying left ventricular (LV) pressure-volume dynamics.As the following experiments show, however, SCS does not alter theseblood parameters—rather, SCS influences and effects the modulation ofneuronal activity within the intrinsic cardiac nervous system. Thus, useof SCS to treat, modify, protect, and influence neuronal activity withinthe intrinsic cardiac nervous system is a novel and non-obvious approachto the pre- and post-treatment of an ischemic heart.

[0257] In the first series of experiments, it is shown that (1) SCSmodifies the capacity of the intrinsic cardiac nervous system togenerate electrical activity; (2) SCS suppresses the excitatory effectsthat local myocardial ischemia exerts on the neurons of the intrinsiccardiac nervous system; and (3) SCS does not change heart indices suchas blood pressure. Thus, the underlying principle that SCS can and doesstimulate and provoke an effect in the intrinsic cardiac nervous systemis shown and demonstrated.

[0258] Electrical stimulation of the dorsal aspect of the upper thoracicspinal cord is used increasingly to treat patients with severe anginapectoris refractory to conventional therapeutic strategies. Clinicalstudies show that spinal cord stimulation (SCS) is a safe adjuncttherapy for cardiac patients, producing anti-anginal as well asanti-ischemic effects. The effects of SCS on the final common integratorof cardiac function, the intrinsic cardiac nervous system, was studiedduring basal states as well as during transient (2 min) myocardialischemia. Activity generated by intrinsic cardiac neurons was recordedin 9 anesthetized dogs in the absence and presence of myocardialischemia before, during and after stimulating the dorsal T1-T2 segmentsof the spinal cord at 66 and 90% of motor threshold using epiduralbipolar electrodes (50 Hz; 0.2 ms; parameters within the therapeuticrange used in humans). The SCS suppressed activity generated byintrinsic cardiac neurons. No concomitant change in monitoredcardiovascular indices was detected. Neuronal activity increased duringtransient ventricular ischemia (46%), as well as during the earlyreperfusion period (68% compared to control). Despite that, activity wassuppressed during both states by SCS.

[0259] Thus, SCS modifies the capacity of intrinsic cardiac neurons togenerate activity. SCS also acts to suppress the excitatory effects thatlocal myocardial ischemia exerts on such neurons. Since no significantchanges in monitored cardiovascular indices were observed during SCS, itis concluded that modulation of the intrinsic cardiac nervous systemmight contribute to the therapeutic effects of SCS in patients withangina pectoris.

Introduction

[0260] Patients who suffer from severe angina pectoris followingcoronary artery revascularization or whose clinical status render theminappropriate candidates for such a procedure can obtain relief fromtheir angina by spinal cord stimulation (SCS) (Jessurun et al., 1997;Schoebel et al., 1997). High frequency, low intensity electrical stimulidelivered to the dorsal aspect of the T₁-T₂ thoracic spinal cordsuppresses the pain associated with myocardial ischemia withoutaffecting awareness of the symptoms from a possible myocardialinfarction (Anderson et al., 1994; Eliasson et al., 1996; Hautvast etal., 1998; Sanderson et al., 1994). Application of SCS does not appearto induce any adverse effects in patients experiencing transientischemia of the myocardium (Sanderson et al., 1992), patients retaintheir capacity to sense angina during increased workload (Mannheimer etal., 1993).

[0261] The effects of SCS have been attributed to improved myocardialperfusion and/or alterations in the oxygen demand and supply ratio asreflected in a reduction in stress-induced alterations in the ST segmentof the ECG (Sanderson et al., 1992). Spinal cord stimulation alsoimproves myocardial lactate metabolism (Mannheimer et al., 1993). Spinalcord stimulation has recently been suggested as an adjunct to coronaryartery bypass surgery in high-risk patients (Mannheimer et al., 1998).

[0262] Spinal cord stimulation has been shown to influence informationprocessing within the central nervous system (Chandler et al., 1193;Yakhnitsa et al., 1999). This treatment modality has also beendemonstrated to influence peripheral blood flow (Augustinsson et al.,1995; Augustinsson et al., 1997; Linderoth et al., 1991; Linderoth etal., 1994; Croom et al., 1997). In order to understand the mechanismsunderlying SCS in cardiac control, we studied the effects of SCS uponthe intrinsic cardiac nervous system. Intrinsic cardiac neurons receiveconstant inputs from spinal cord neurons to regulate regional cardiacfunction on a beat-to-beat basis (NAMES). Transient regional ventricularischemia markedly increases the activity generated by intrinsic cardiacneurons (Huang et al., 1993). Furthermore, excessive activation oflimited populations of intrinsic cardiac neurons induces cardiacdysrhythmias, even in normally perfused hearts (Huang et al., 1994).

[0263] The experiments and data detailed hereinbelow show that SCS,applied with clinically employed electrical stimulation parameters,modifies the activity generated by intrinsic cardiac neurons in situ.SCS does not change cardiac dynamics. Effects of SCS on intrinsiccardiac neural activity were characterized during coronary arterialocclusion as well as during the subsequent reperfusion period and it wasdetermined that SCS modifies intrinsic cardiac neuronal function in thepresence of myocardial ischemia. These experiments show that SCSinfluences the behavior of intrinsic cardiac neurons markedly, changesthat are involved in the clinically observed effects of SCS during acutemyocardial ischemia.

METHODS

[0264] Animal Preparation

[0265] Experiments performed in the present study were approved by theInstitutional Animal Care and Use Committee of the OUHSC and followedthe guidelines outlined by the International Association for the Studyof Pain and in the NIH Guide for the Care and Use of Laboratory Animals(National Academy Press, Washington, DC, 1996). Nine adult male dogs ofmixed breed weighing between 15 and 25 kg were used. Animals were keptunder standard laboratory conditions in a light-cycled environment (12h/12 h) with free access to water at all times and to food at regularintervals. For the duration of the surgery, dogs were first anesthetizedwith sodium thiopental (20 mg/kg, i.v.) and maintained with sodiumthiopental administered in boluses (5 mg/kg i.v.) to effect every 5-10min. Animals were intubated and then artificially ventilated using aHarvard respirator (Palm Springs, Calif.). After the surgicalpreparation was completed, anesthesia was changed to alpha chloralose.An initial bolus dose of alpha chloralose (75 mg/kg, i.v.) wasadministered, with repeat doses (20 mg/kg) given as required during theremainder of the experiment. The level of anesthesia was checkedthroughout each experiment by observing pupil reaction, monitoring jawtension and squeezing a hindpaw to determine if blood pressure and heartrate changed. This anesthetic regimen has been demonstrated to produceadequate anesthesia without suppressing autonomic neural responses(Gagliardi et al., 1988). Electrodes inserted into the forelimbs and theleft hind limb were connected to an Astro-Med, Inc. (West Warwick, R.I.)model MT 9500 eight channel rectilinear recorder to monitor a modifiedLead II electrocardiogram.

[0266] Implantation of Spinal Cord Stimulation Electrodes

[0267] After induction of anesthesia, animals were placed in the proneposition and the epidural space of the mid-thoracic spinal column waspenetrated percutaneously with a Touhy needle using A-P fluoroscopy andloss-of-resistance technique, as is routinely done in the clinic. Afour-pole catheter (Medtronic QUAD Plus Model 3888; Medtronic Inc.,Minneapolis, Minn.) was introduced through the cannula and its tip wasadvanced to the T₁ level of the spinal column and placed slightly to theleft of the midline (Augustinsson et al., 1995). The two poles of thisstimulating lead chosen for subsequent use (inter-electrode distance of1.5 cm) were placed at the level of the T₁ and T₄ vertebrae. Finalplacement was aided by delivering electrical current to induce motorresponses using the rostral or caudal poles as cathodes, respectively.Rostral stimulation just above motor threshold resulted in proximalforepaw and/or shoulder muscle contractions while caudal electrodestimulation induced contractions in the lower trunk. Once theappropriate electrode positions were obtained, the lead was fixed to theintraspinous ligaments with a suture surrounding a Silicone protectivesleeve. Extension wires were tunneled subcutaneously to the ventralsurface of the animal where they were connected to a stimulator. Motorresponses were rechecked after the animal had been turned to the supineposition to make sure the electrodes had not moved during this maneuverand to establish the appropriate stimulus intensities for the subsequentSCS.

[0268] Cardiac Instrumentation

[0269] After placing the animal on its back, a bilateral thoracotomy wasmade in the fifth intercostal space to expose the heart. The subclavianansae on both sides of the thorax were exposed and silk ligatures wereplaced around them so that each could be easily sectioned later in theexperiments to decentralize the intrinsic cardiac nervous system. Theventral pericardium was incised and retracted laterally to expose theheart and the ventral right atrial deposit of fat containing the ventralcomponent of the right atrial ganglionated plexus. Neurons in thisganglionated plexus are representative of those found in the variousintrinsic cardiac ganglionated plexuses (Gagliardi et al., 1988).

[0270] Left atrial chamber pressure was measured via a PE-50 catheterinserted directly into the left atrial chamber via its appendage. Leftventricular chamber pressure was monitored via a Cordis (Miami, Fla.) #6French pigtail catheter, which was inserted into that chamber through afemoral artery. Systemic arterial pressure was measured using a Cordis#7 French catheter placed in the descending aorta via the other femoralartery. These catheters were attached to Bentley (Irvine, Calif.)Trantec model 800 transducers.

[0271] Neuronal Recording

[0272] Activity generated by ventral right atrial neurons was recordedin situ, as has been done in previous studies (Gagliardi et al., 1988).To minimize epicardial motion during each cardiac beat, a circular ringof stiff wire was placed gently on the fatty epicardial tissue overlyingthe ventral surface of the right atrium containing the right atrialganglionated plexus. A tungsten microelectrode (30-40 μm diameter andexposed tip of 1 μm; impedance of 9-11 M at 1000 Hz), mounted on amicromanipulator, was lowered into this fat using a microdrive.Exploration was done by driving the electrode tip through this tissuebeginning at the surface of this fat, penetrating to regions adjacent tocardiac musculature. Proximity to the atrial musculature was indicatedby increases in the amplitude of the ECG artifact. The indifferentelectrode was attached to mediastinal connective tissue adjacent to theheart. Signals recorded via the electrode were led to a CWE BMA-831differential preamplifier with a high impedance head stage (bandpassfilters set at 300 Hz and 10 kHz), and were processed by a signalconditioner (bandpass 100 Hz-2 kHz). Signals were amplified further viaa Princeton Applied Research (Princeton, N.J.) battery driven amplifier(300 Hz-2 kHz) and were displayed on an Astro-Med, Inc. (West Warwick,R.I.) MT 9500 8 channel rectilinear recorder along with thecardiovascular variables described above. Data were stored via a Vetter(Rebesburg, Pa.) M3000A digital tape system for later analysis. Actionpotentials generated by neurons in one site of a right atrialganglionated plexus were recorded using extracellular recordingelectrodes, individual units being identified by their amplitudes andconfigurations. As established previously (Armour et al., 1990),extracellular action potentials so generated are derived from somataand/or dendrites rather than axons of passage. Amplitudes of identifiedaction potentials varied by less than 25 μV over several minutes. Eachpotential retained the same configuration over time. Action potentialsrecorded in a given locus with the same configuration and amplitude(±25μV) were considered to be generated by a single unit.

PROTOCOLS

[0273] Five different protocols were employed in each animal (cf. FIG.34) The order in which each protocol was applied was randomized amonganimals.

[0274] Protocol A-spinal Cord Stimulation

[0275] The parameters used to electrically stimulate the thoracic spinalcord were similar to those used clinically. Stimuli were delivered tothe dorsal aspect of the thoracic spinal cord via a Grass model S48stimulator connected to the quadripolar electrode via a stimulusisolation unit (Grass model CCU1) via, a constant current unit (GrassSIU1). With the animal placed in the supine position for all subsequentexperimentation, the current intensity used to evoke detectable skeletalmuscle motor responses was determined as the motor threshold (MT).Stimuli (50 Hz and 0.2 ms duration) were delivered at two intensities(66 and 90% of MT). An intensity of 66% of MT has been shown to recruitlow threshold, rapidly conducting axons (A-beta), whereas higher,intensity stimuli (90%) activate fast A-delta fibers as well as theother axonal populations (Croom et al., 1997; Linderoth et al., 1991).The current measured at MT varied among animals likely because of thevaried anatomy of the thoracic spinal space among animals. The stimulusintensity was found to vary between 30 and 50 μA when current was set to66% of MT. When the stimulus current was 90% of MT, it varied between 80and 210 μA among different animals. The MT was rechecked periodicallyand remained stable over time in individual animals. With respect toprotocol A, cardiac indices and intrinsic cardiac neural activity weremonitored immediately before, during and for 30-45 s after 4 ruin of SCSat 90% of MT (FIG. 34 (A)).

[0276] Protocol B-regional Ventricular Ischemia

[0277] A silk (3-0) ligature was placed around the left anteriordescending coronary artery and another around the circumflex coronaryartery, approximately 1 cm from their respective origins. Each ligaturewas led through a short segment of polyethylene tubing in order toocclude these arteries later in the experiments while leaving thearterial blood supply (right coronary and sino-atrial arteries) patentto the ventral right atrial neurons that were being investigated. Furprotocol B, cardiac indices and neuronal activity were monitored before,during and immediately after occluding the two coronary arteriesconcurrently for 2 min (FIG. 34(B)).

[0278] Protocols C, D and E in which SCS and Regional VentricularIschemia were Combined

[0279] The effects of 2 min of myocardial ischemia on intrinsic cardiacneuronal activity and regional cardiac indices were studied in thepresence of SCS (at 90% of MT for 4 min) applied at different timesduring the myocardial ischemia. Protocol C: The spinal cord wasstimulated for 4 min and the 2 min of coronary artery occlusion began 1min after the onset of the SCS (in the middle of the SCS; Foreman FIG.1C). Protocol D: Spinal cord stimulation was initiated 1 min aftercoronary artery occlusion began (staged occlusion with overlappingstimulation) (FIG. 34(D)). Protocol E: In this protocol, spinal cordstimulation began immediately after finishing 2 min of coronary arteryocclusion (FIG. 34(E)). The order in which each of these protocols wasapplied was randomized among dogs.

[0280] After all of the protocols described above were completed, theright and left subclavian ansae were sectioned in five of the dogs,thereby eliminating spinal cord afferent and efferent communicationswith neurons in intrathoracic ganglia. After this maneuver, the five SCSand transient coronary occlusion protocols described above wererepeated.

DATA ANALYSIS

[0281] Individual action potentials, which maintained theirconfigurations over time, were analyzed. Activity generated by thesomata and/or dendrites of neurons within the right atrial ganglionatedplexus was averaged during successive 30-s periods before, during andafter each intervention. At the same time, heart rate, left ventricularwall (intramyocardial) and chamber systolic pressures were measured, aswas aortic pressure. Neuronal activity and cardiovascular indicesrecorded immediately before each intervention and during the steadystate response to an intervention were averaged and presented as means±S.E.M. Fluctuations in the amplitude of action potentials generated bya unit varied by less than 50 μV over several minutes, action potentialsretaining the same configurations over time. Thus, action potentialsrecorded in a given locus with the same configuration and amplitude (±50μV) were considered to be generated by a single unit. Action potentialswith signal-to-noise ratios greater than 3:1 were analyzed. Thethreshold for neuronal activity changes was taken as a change of morethan 20% from baseline values. Neuronal activity responses elicited byeach intervention were evaluated by comparing activity generatedimmediately before each intervention with data obtained at the point ofmaximum change during the intervention. Data were expressed as means±S.E.M. Oneway ANOVA and paired t-test with Bonferroni correction formultiple tests were used for statistical analysis. A significance valueof P<0.05 was used for these determinations.

RESULTS

[0282] Identification of Active Sites

[0283] Action potentials were identified in 1-3 loci within the ventralright atrial ganglionated plexus of each animal. Based on the differentamplitudes and configurations of the recorded action potentials withinthese loci, ongoing activity was generated by an average of 5.1±0.9(range 3-9) neurons. Identified neurons generated, on average, 496±112impulses per minute (ipm) during control conditions throughout theduration of these experiments. Multiple neurons at each identifiedactive site generated action potentials that were altered in a similarfashion by each of the different interventions tested.

[0284] (Protocol A) Effects of Spinal Cord Stimulation

[0285] Only the effects of SCS employed at 90% of MT are presentedherein since 66% MT elicited minimal changes in the activity generatedby the intrinsic cardiac neurons. The average activity generated byidentified right atrial neurons in all animals (n=9) fell from 496±112to 1501±71 ipm (P<0.01) during SCS at 90% MT (FIG. 34(A)). Neuronalactivity remained depressed for 10-20 s after SCS ceased (142±61 ipm),returning to control levels by about 1 min after cessation ofstimulation (FIG. 35(A)). SCS did not change monitored cardiac indicesoverall. For instance, SCS did not change heart rate (155±8 vs. 159±8beats per minute) or left ventricular chamber systolic 124±8 vs. 131±8mmHg) and diastolic pressures. SCS did not change aortic pressure(124±8/99±6 vs. 122±5/95±4 mmHg).

[0286]FIG. 35. shows initiation of coronary artery occlusion (arrowbelow) resulted in an increase in the activity generated by right atrialneurons (individual units identified by action potentials greater thanthe small atrial electrogram artifacts). From above down are the ECG,aortic pressure (AP), left ventricular chamber pressure (LVP) andneuronal activity. Horizontal timing bar=30 s.

[0287] (Protocol B) Effects of Transient Myodcardial Ischemia

[0288] When ventricular ischemia was induced by occluding both the leftanterior descending and circumflex coronary arteries for 2 min inneurally intact preparations, the activity generated by right atrialneurons increased in each animal (FIG. 35). Neuronal activity increased,on average, by 46% (370±126 to 539±91 ipm; P<0.01) (FIGS. 35(B) and CC)despite the fact that the blood supply of identified neurons wasunaffected. Neuronal activity remained elevated immediately afterreperfusion began (621±175 ipm, +68% compared to control values;P<0.05). Monitored cardiac indices did not change significantly duringthe 2 min of coronary artery occlusion or during the reperfusion period.For instance, heart rate was similar at the end of ventricular ischemiaepisodes (140±10 beats per minute) as before these episodes began (142±9beats per minute). Even though left ventricular chamber systolicpressure underwent minor reductions in a few instances, this indexremained unchanged overall (121±6 vs. 121±6 mmHg). Left ventriculardiastolic pressure and aortic pressure were also unaffected. No seriousdysrhythmias were induced during the brief periods of coronary arteryocclusion. During the early reperfusion phase, minor S-T segmentelevation and terminal QRS slurring was evident in each animal.

[0289] SCS Modulated Responses to Transient Myocardial Ischemia

[0290] Neuronal activity was not enhanced by coronary artery occlusioninduced in the presence of SCS, irrespective of whether SCS was appliedduring (FIGS. 34c and 34(D)) or immediately after (Foreman FIG. 1E) theischemic period. Monitored cardiovascular variables did not changesignificantly when the combined coronary artery occlusion and SCSprotocols were instituted.

[0291] (Protocol C) Occlusion in the Middle of Stimulation

[0292] When the 2-min period of myocardial ischemia occurred in themiddle of the SCS (1 min after SCS began), the neurosuppressor effectsof SCS persisted during the ischemic period (FIG. 36). For instance,intrinsic cardiac neuronal activity was reduced from that of controlstates (511±197 ipm) during SCS (169±99 ipm, P<0.01 compared tocontrol), neuronal activity remaining suppressed when the stimulationoccurred in conjunction with the occlusion (164±74, P<0.01 compared tocontrol; FIGS. 34-C). Suppression of neuronal activity persisted afterterminating the occlusion while the SCS was maintained (166±84 ipm,P<0.01 compared to control). Only after discontinuing the SCS didneuronal activity gradually return to control values.

[0293] (Protocol D) Occlusion Overlapped by Stimulation

[0294] During this protocol (FIG. 34(D)), the activity generated byintrinsic cardiac neuronal activity was enhanced by 42% (388±155 to555±211 ipm; P<0.01) during the initial coronary artery occlusionperiod. When SCS was applied 1 min after the occlusion began, neuronalactivity was suppressed by 46% (activity of 211±134 ipm) even though themyocardial ischemia persisted (FIGS. CC-C). In this protocol, neuronalactivity remained suppressed during the reperfusion period (227±134 ipm)while the SCS persisted neuronal activity returned to control valuesonly after SCS ceased (394±142 ipm).

[0295]FIG. 36 shows the influence of SCS on the ECG, left ventricularchamber pressure (LVP=145 mmHg) and intrinsic cardiac neuronal activity(lowest line) before and during coronary artery occlusion. (A) Multipleneurons generated action potentials, represented by their differingheights, at a rate of 132 impulses per minute (ipm) during controlstates. (B) Once SCS was initiated (note stimulus artifacts in theneuronal tracing), neuronal activity decreased to 34 imps/min (noactivity generated during the record). ECG alterations were inducedthereby. (C) Neuronal activity continued at that rate (39 ipm) in thepresence of SCS even though coronary artery occlusion had beenmaintained for over 1.5 min.

[0296] (Protocol E) Occlusion Followed by Stimulation

[0297] In this protocol (FIG. 34(E)), coronary artery occlusion aloneenhanced neuronal activity (403±150 to 701±315 ipm; P<0.01). When SCSwas started immediately following termination of 2 min of coronaryocclusion (that is during the early reperfusion period), neuronalactivity fell to 173±295 ipm (P<0.01 compared to the ischemia period).Neuronal activity remained suppressed throughout this stimulationperiod, being 244±98 ipm (P<0.01 compared to control values) after 4 minof SCS. This is in distinct contrast to the finding that neuronalactivity remained elevated (˜70% of control values) during the earlyreperfusion period immediately after SCS was terminated (FIG. 34(B)).

[0298] Acute Decentralization

[0299] After all of the experimental protocols described above werecompleted, the spinal cord was stimulated in 5 animals at 90% of MTbefore and after sectioning the right and left ventral and dorsalsubclavian ansae. After surgically disconnecting intrinsic cardiacneurons from the spinal cord neurons, ongoing neuronal activitydecreased from 378±34 to 162±72 ipm (P<0.01). SCS did not modify theactivity generated by identified intrinsic cardiac neurons thereafter(162±72 vs. 147±61 ipm); nor did SCS affect recorded cardiac indices.

Discussion

[0300] Results of the present experiments demonstrate that the activitygenerated by intrinsic cardiac neurons is modulated when the dorsalaspect of the thoracic spinal is stimulated electrically. Thatsuppression of the ongoing activity generated by intrinsic cardiacneurons induced by SCS persisted for at least 30 s following terminationof 4 min of SCS shows that the effects of this intervention last beyondthe stimulation period. Interruption of afferent and efferent nervestraveling in the subclavian ansae eliminated the suppressor effects thatSCS exerted on intrinsic cardiac neurons. These data show that theinfluence of spinal cord neurons on the intrinsic cardiac nervous systemoccur primarily via axons coursing in the intrathoracic sympatheticnervous system.

[0301] Based on results obtained when SCS was applied to the lumbosacralspinal cord, both sympathetic afferent and efferent fibers contribute tothe suppression of intrinsic cardiac activity so identified. Fourminutes of SCS at 66% of MT was much less effective in suppressingneuronal activity than when the spinal cord was stimulated at 90% of MT.Spinal cord stimulation at 90% MT antidromically activates sensoryafferent fibers that release calcitonin gene-related peptide (CGRP) fromtheir afferent terminals, an action that may be dependent on thepresence of nitric oxide; such local release of CGRP from sensoryafferent nerve terminals produces vasodilation of the rat hind paw(Croom et al., 1997). It is known that endorphins are released into thecoronary circulation of humans during SCS (Eliasson et al., 1991). Therelease of neuropeptides by antidromic activation of sensory neurites(Croom et al., 1997) acts to change the activity generated by intrinsiccardiac neurons (Armour et al., 1993).

[0302] Activation of sympathetic efferent preganglionic axons suppressesmany intrathoracic reflexes that are involved in cardiac regulation(Armour et al., 1985) as well as the activity generated by populationsof neurons within intrathoracic extracardiac (Armour, 1986) andintrinsic cardiac (NAMES) ganglia, thereby reducing the capacity ofintrathoracic sympathetic efferent neurons to influence cardiodynamics(Butler et al., 1988). This effect may in part be due to activatinginhibitory synapses within intrathoracic ganglia, including those on theheart such as occurs when intracranial pressure raises (Murphy et al.,1995). Such suppression of neuronal activity has been demonstrated insympathetic efferent neurons controlling the peripheral vasculature aswell (Linderoth et al., 1991; Linderoth, Fedorcsak et al., 1991).

[0303] As has been shown previously (Huang et al., 1993), the activitygenerated by right atrial neurons increased in the presence of regionalventricular ischemia (FIG. 35), remaining elevated during the earlyreperfusion phase (FIG. 34(B)). This information and knowledge hasclinical relevance since excessive activation of limited populations ofintrinsic cardiac neurons can lead to the induction of ventriculararrhythmias (Huang et al., 1994) or even ventricular fibrillation(Armour, 1999). Application of SCS before and during the induction oftransient coronary artery occlusion prevented ischemia-induced changesin neuronal activity (FIG. 36), including that identified during thereperfusion period (FIG. 34(C)). In other words, although intrinsiccardiac neuronal activity was enhanced during regional ventricularischemia, SCS returned intrinsic cardiac neuronal activity to base linelevels during these ischemic episodes. It is important to note that thetwo coronary arteries that were occluded did not supply arterial bloodto identified right atrial ventral neurons (Huang et al., 1993). Thetransient periods of regional ventricular ischemia were of short enoughduration to induce minor or no alterations in recorded cardiacvariables. Thus, the effects of transient coronary artery occlusion onintrinsic cardiac neuronal activity are the result of altered inputs tointrinsic cardiac neurons arising from distant ischemia-sensitiveafferent neurites. SCS was effective in reducing such inputs. Theseneurosuppressor effects occurred whether SCS was applied immediatelybefore or during coronary artery occlusion, or during the earlyreperfusion phase (FIG. 34). These data support the notion that SCSsuppresses intrinsic cardiac neurons responsiveness to regionalventricular ischemia as well as during the subsequent reperfusionperiod.

[0304] The data obtained in this study are in accord with clinicalfindings indicating that improvement of cardiac function and symptomscan occur when SCS is applied to patients with angina pectoris(deJongste et al., 1994). Since modification of the intrinsic cardiacnervous system can lead to alterations in ventricular regional flow(Kingma et al., 1994), perhaps some of the responses elicited by SCSinvolved subtle changes in the redistribution of coronary artery bloodflow given that no detectable changes in cardiodynamics were identifiedwith the methods used in these experiments. Thus, the effects that SCSinduces in a clinical setting resides, in part, in the capacity of suchtherapy to stabilize this final common regulator, even in the presenceof ventricular ischemia. Since the intrinsic cardiac nervous systemreceives inputs arising from cardiac sensory nenrites as well as fromcentral neurons (Murphy et al., 1995), SCS may exert multiple effects onthis local neuronal circuitry. Heterogeneous activation of intrinsiccardiac neurons destabilize cardiac neuronal regulation that, in turn,leads to the genesis of ventricular tachydysrhythmias (Huang et al.,1994). Data obtained in the present experiments indicated that SCSreduces the excitability of intrinsic cardiac neurons, even in thepresence of ventricular ischemia and, as such, may help to stabilizecardiac function.

[0305] In summary, electrical stimulation of the thoracic spinal cordinfluences the function of the final common neuronal regulator ofcardiac function, the intrinsic cardiac nervous system, even in thepresence of myocardial ischemic challenge. Thus, SCS acts in part toprotect the heart from some of the deleterious consequences resultingfrom myocardial ischemia via altering the function of the intrinsiccardiac nervous system.

[0306] As stated previously, electrical stimulation of the dorsal aspectof the upper thoracic spinal cord is used to treat patients with anginapectoris refractory to conventional therapeutic strategies. The purposeof the following described experiments was to determine whether spinalcord stimulation SCS in dogs affects regional myocardial blood flow andleft-ventricular LV function before and during transient obstruction ofthe left anterior descending coronary artery LAD. In anesthetized dogs,regional myocardial blood flow distribution was determined usingradiolabeled microspheres and left-ventricular function was measured byimpedance-derived pressure-volume loops. SCS was accomplished bystimulating the dorsal T1-T2 segments of the spinal cord using epiduralbipolar electrodes at 90% of motor threshold MT 50 Hz, 0.2-ms duration.Effects of 5-min SCS were assessed under basal conditions and during4-min occlusion of the LAD.

[0307] In summary, SCS alone evoked no change in regional myocardialblood flow or cardiovascular indices. Transient LAD occlusionsignificantly diminished blood flow within ischemic, but not innon-ischemic myocardial tissue. Left ventricular pressure-volume loopswere shifted rightward during LAD occlusion. Cardiac indices werealtered similarly during LAD occlusion and concurrent SCS. Thus, SCSdoes not influence the distribution of blood flow within thenon-ischemic or ischemic myocardium. Nor does it modify LVpressure-volume dynamics in the anesthetized experimental preparation.

INTRODUCTION

[0308] The majority of patients with angina pectoris secondary tocoronary artery disease can be adequately controlled with medication andrevascularization procedures. However, a subset of patients exists withchronic angina that is refractory to these standard treatmentstrategies. Neuromodulation therapy has been advocated as an adjuncttherapy for patients with chronic refractory angina pectoris (DeJongste,Hautvast et al., 1994; Mannheimer et al., 1985) or even as analternative to coronary artery bypass grafting CABG in high-riskpatients (Mannheimer et al., 1998) to improve the clinical response toischemia. With regard to safety concerns, electrical stimulation of thedorsal aspect of the spinal cord SCS does not mask anginal symptomselicited during acute myocardial infarction (Anderson et al., 1994;Sanderson et al., 1994). Furthermore, SCS appears to have anti-ischemicproperties as demonstrated during exercise stress testing (DeJongste,Hautvast et al., 1994; Sanderson et al., 1994; Hautvast et al., 1994),ambulatory ECG monitoring (DeJongste et al., 1994; Hautvast et al.,1997), and rapid right atrial pacing (Mannheimer et al, 1993; Sandersonet al., 1994).

[0309] Chauhan et al. 1994 showed that the velocity of coronary arterialblood flow of patients with either CAD measured in the left main arterywith stenosis >50% in the right coronary artery or syndrome X changedwhen transcutaneous electrical nerve stimulation TENS; 150 Hz at 300 ms,10-60 mA is applied for 5 min. In agreement with that, other reportsindicate that SCS can increase myocardial blood flow in low-flowregions, possibly related to recruitment of coronary collateral vesselsand a decrease in flow in normally perfused myocardium (Mobilia et al.,1998). Other contrary studies indicate, however, that SCS does notimprove blood flow within the ischemic myocardium of patients withsignificant coronary artery disease or syndrome X even though it reducesST-segment alterations (De Landesheere et al., 1992; Sanderson et al.,1996; Jessurun et al., 1998; Norrsell et al., 1998). Furthermore, atleast one study has indicated that SCS does not alter total coronaryblood flow in patients undergoing dipyramidole stress testing (Hautvastet al., 1996).

[0310] In light of these divergent clinical findings, examination of theinfluence of SCS on the distribution of regional myocardial blood flowutilizing radiolabeled microsphere technique (Baer et al., 1984) and LVchamber dynamics utilizing the conductance catheter technique (Baan etal., 1984) in canine hearts was undertaken. As has been indicated in thepreviously discussed experiments hereinabove, SCS does not alter cardiacindices (Foreman et al., 2000). Thus, the effects of altered cardiacworkload on regional ventricular flow elicited during SCS was expectedto be minimal. For that reason, the effects of SCS on the distributionof blood flow in the acutely ischemic myocardium was also examined. Theduration of ischemic period was brief in order that cardiac indicesreturn to normal values after terminating regional myocardial ischemia.The results obtained from these disclosed experiments indicate that SCSdoes not affect regional myocardial blood supply nor LV dynamics in thenormally perfused myocardium. Furthermore, SCS does not alter blood flowwithin a ventricular ischemic zone; nor does it affect theischemia-induced rightward shift of the LV pressure-volume relationship.

MATERIALS AND METHODS

[0311] Animal Preparation

[0312] The experiments performed in the present study were performed inaccordance with the Guide to the Care and Use of Experimental Animalsset up by the Canadian Council on Animal Care and under the regulationsof the Animal Care Committee at Laval University. Adult mongrel dogs ofeither sex, weighing between 20 and 25 kg, were used. Dogs weretranquilized with diazepam 1 mg/kg, i.v. and fentanyl 20 mg/kg, i.v. andthen anesthetized with sodium pentobarbital 25 mg/kg, i.v. Noxiousstimuli were applied occasionally to a paw throughout the experiments toascertain the adequacy of the anesthesia. Repeat doses of pentobarbital5 mg/kg, i.v. were administered throughout the experiments as required.Dogs were intubated and mechanically ventilated with a mixture of oxygen25% and room air 75% , maintaining an end-expiratory pressure of 5-7 cmH₂O to prevent atelectasis. Respiratory rate and tidal volume wereadjusted to maintain arterial blood gases within physiological values.Body temperature was monitored and kept between 37.58C. and 38.58C. by awater-jacketed Micro-Temp heating unit Zimmer, Dover, Ohio, USA.

[0313] Spinal cord Stimulation

[0314] Implantation of the Spinal Cord Stimulation Electrodes

[0315] After the animal was placed in the prone position, the epiduralspace was entered with a Touhy needle via a small skin incision in thelower thoracic region. A four-pole lead Medtronic QUAD Plus Model 3888;Medtronic, Minneapolis, Minn. was advanced rostrally in the epiduralspace to the upper thoracic level under anterior-posterior fluoroscopyand positioned slightly to the left of midline according to currentclinical practice (DeJongste et al., 1994). The most cranial pole of thelead was positioned at the T1 level. Electrical current was deliveredvia the rostral and caudal poles to verify their functional positioning.Increasing stimulus intensity via the rostral pole as cathode to motorthreshold intensity MT-induced muscle contractions in the proximalforepaw and shoulder. Stimulation with the caudal pole as cathode at MTactivated thoracic paravertebral muscles, resulting in a twistingmovement of the trunk. When a satisfactory electrode position wasobtained, the lead, protected by a silicon sleeve, was fixed to theinterspinous ligament and then connected to an external stimulator.

[0316] Threshold Determination for Spinal Cord Stimulation

[0317] The animal was shifted to the decubitus position for theremainder of the experiment and MT was then reestablished. SCS wasdelivered via the indwelling electrode connected to a Grass S48Stimulator Grass Instruments, Quincy, Mass., USA via a stimulusisolation unit Grass SIU 5B and a constant current generatorGrassrCCU1A. The parameters used to stimulate the spinal cord were 50 Hzand 0.2-ms duration; these values are the same as those used previouslyto reduce neuronal activity of intrinsic cardiac neurons in anesthetizeddogs (Foreman et al., 2000). Stimulation intensity was 90% of thatevoking a motor response and corresponds to the maximum used in patients(Chandler et al., 1993; Anderson et al., 1994). The current intensityused for SCS at 90% of MT, varied between 0.16 and 0.72 mA mean: 0.44 mAamong animals. The most rostral and caudal poles were chosen as cathodeand anode, respectively, so that the entire spinal cord area used forangina therapy in humans would be stimulated.

[0318] CardioVascular Instrumentation

[0319] Both femoral arteries and the right femoral vein were exposed andcannulated with 8F vascular introducers. Cordis, Miami, Fla., USA. A 7F12-electrode conductance catheter with Pigtail and vascular port Cordis,Roden, The Netherlands was advanced into the LV chamber via the leftfemoral artery. Pressure transducers were connected to the vascular portof the conductance catheter and to a fluid-filled catheter Cordis a4placed in the descending aorta. A femoral vein catheter was used forperiodic drug injections and for fluid replacement therapy physiologicalsaline. Surface needle electrodes were positioned to record a standardlead II electrocardiogram. Analog data were displayed on an Astro Medmodel MT-9500 polygraph and stored directly on a computer hard disk at asampling rate of 333 samples/channel using the AxoScope data acquisitionsoftware Axon Instruments, Foster City, Calif., USA . Total LVpressure-volume loops were determined from changes in the electricalimpedance measured by the summed volumes using a signalconditioner-processor Leycom Model Sigma-5, Oegstgeest, The Netherlands, as described previously (Baan et al., 1984). A computer analysissystem Conduct-PC, Cardiodynamics, Leiden, The Netherlands was used toassess LV pressure-volume loops.

[0320] Placement of Coronary Artery Occluder

[0321] Under fluoroscopy, a modified right Judkins catheter 8F, Cordis,USA was advanced to the left coronary ostium. Thereafter, a ballooncatheter was advanced into the left anterior descending LAD coronaryartery. The position of the balloon catheter was verified by injectionof contrast medium Hexabrix 320, Malinckrodt Medical, Pointe-Claire, CANinto the left main coronary artery, visualized in the left anterioroblique position. A baseline coronary artery angiogram was obtained toconfirm positioning of the balloon in the ventral descending coronaryartery about 2 cm from its origin.

[0322] Experimental Protocol

[0323] Surgical preparation and angiographic balloon catheter placementwere followed by a 30-min stabilization period. Regional blood flow andLV dynamics were obtained at: 1 baseline C1 ; 2 during 5-min SCS; 3return to steady-state conditions C2 ; 4 4 min of LAD occlusion CO; 5return to steady-state conditions C3 ; and 6 5-min SCS during which timeblood flow in the LAD was stopped for 4 minutes. The experimentalprotocol was always begun with interventions 1 and 2 since the initialgoal was to assess the effects of SCS on myocardial blood supply and LVdynamics. SCS and coronary occlusion were performed twice in eachanimal. Four dogs underwent the following protocol sequencebaseline-SCS; baseline-LAD occlusion; baseline-SCS/LAD occlusion . Inanother four dogs, the protocol sequence was altered baseline-SCS;baseline-SCS/LAD occlusion; baseline-LAD occlusion . Blood flow in theLAD was totally obstructed by inflating the angiocatheter balloon ns8 toa pressure of 8 atm Inde-flator Plus 20, ACS, Tomecula, Calif., USA for4 min. Completeness of coronary obstruction was confirmed by injectionof contrast medium under fluoroscopy. At least 10 min elapsed betweeneach intervention to stabilize the experimental preparation. The timeduring which the coronary artery was occluded 4 min was of sufficientduration to alter regional dynamics cf., the pressure-volumerelationship, yet result in a return to control values upon restorationof coronary artery blood flow.

[0324] Measurement of Regional Myocardial Blood Flow

[0325] Regional blood flow distribution was determined using theradioactive microsphere technique (Baer et al., 1984). Six differentradiolabeled microspheres Sn, Sr, Nb, Sc, Ce, In, each with a diameterof 15 mm, were obtained from NEN Boston, Mass., USA . Immediately priorto injection, the microsphere suspension was agitated in a vortex mixerfor 2 min. Each injection comprised 1.6−3=106 microspheres administeredinto the LV chamber as a bolus over 15-20 s and flushed with 15 ml ofwarmed saline. For each microsphere injection, a timed collection ofarterial blood was performed with a Masterflex infusion/withdrawal pumpFisher, Montreal, CAN from the right femoral artery catheter at aconstant rate of 7.5 ml/min beginning 10 s before microsphere injectionand continuing for 2 min. Myocardial blood flow was evaluated in alldogs at six different time points: 1 during baseline state before anyintervention had commenced control, 2 during the final 2 min of the5-min SCS period, 3 baseline control a2 i.e., 10 min after return tobaseline conditions, 4 at the midpoint of coronary occlusion, 5 baselinecontrol a3 i.e., 10 min after return to baseline conditions , and 6during the final 2 min of the 5-min SCS plus 4-min coronary occlusionperiod.

[0326] Anatomic Risk Zone Analysis

[0327] At the end of each study, the angiographic balloon catheter wasre-inflated; contrast medium was injected to verify that the balloon waspositioned in the same location used earlier to induce regionalmyocardial ischemia. Monastral blue dye 5 ml was injected directly intothe coronary artery distal to the occlusion site to identify theischemic zone. During deep pentobarbital sodium anesthesia, cardiacarrest was induced by intravenous injection of saturated potassiumchloride. The heart and left kidney were excised rapidly from the body,rinsed in saline at room temperature and then fixed in 10% bufferedformaldehyde. For blood flow analysis, the right ventricle was removedand the LV including interventricular septum was cut into 6-mm slicesfrom apex to base parallel to the atrioventricular groove. Fourtransverse myocardial sections beginning with the second most apicalslice were employed for blood flow analysis. The LV was divided intoanterior ischemic and posterior non-ischemic segments and furthersubdivided into endocardial, midmyocardial and epicardial portions. Theoutlines of each LV slice, cavity area and the area at risk i.e.,containing blue dye were traced onto acetate sheets.

[0328] Planimetry with Sigma Scan software; SPSS, California, USA wasperformed on these using a digitizing tablet Summagraphics II Plusinterfaced with a personal computer to determine respective surfaceareas. The results so obtained were expressed as the area at riskindexed to total left-ventricular mass. Regional blood flow was alsoassessed in 4 kidney slices excluding the most polar slice that werefurther subdivided into medulla and cortex regions. Radioactivity in alltissue and blood reference samples was measured in a gamma-wellscintillation counter Cobra. II, Canberra Packard Instruments, Montreal,CAN with standard window settings. Tissue counts were corrected forbackground, decay and isotope spillover; regional blood flow ml/min/gwas calculated using the PCGERDA computer software Packard Instrumentsand expressed in Ml/min/g.

DATA ANALYSIS

[0329] Heart rate, arterial pressure, LV pressure and LV pressure-volumeloops were evaluated on a beat-to-beat basis and averaged for 30 s priorto and during each intervention. Comparisons of cardiac hemodynamics anddistribution of myocardial blood flow during different experimentalconditions was performed using analysis of variance ANOVA with repeatedmeasures. When a significant effect of treatment was obtained, pair wisecomparisons were made using Scheffe's post-hoc test. All statisticalprocedures were performed using the SAS statistical software packageSAS, Cary, N.C., USA ; a pFO.05 was considered significant.

RESULTS

[0330] Ten dogs entered into the study; two dogs one during AD occlusionand one during LAD occlusion with con-current SCS went into intractableventricular fibrillation and were excluded from the data analysis.

[0331] CardioVascular Variables

[0332] Heart rate, LV end-systolic and end-diastolic pressures and meanaortic pressure did not change during SCS. Table III LV stroke volumeand ejection fraction were likewise unaffected by SCS. Monitoredcardiovascular indices did not change significantly during 4 min of LADocclusion Table III. TABLE III Summary of cardiac hemodynamics HRLVP_(sys) LVP_(dias) PaoM RPP ESV EDV SV C1 107 ± 12 71 ± 3 2 ± 1 64 ± 47.29 ± 0.80 33.8 ± 2.7 39.7 ± 2.6 7.7 ± 1.1 CS 110 ± 12 72 ± 4 1 ± 1 63± 4 7.60 ± 0.83 33.3 ± 2.3 39.5 ± 2.7 7.8 ± 2.1 C2 103 ± 16 70 ± 2 1 ± 164 ± 3 7.10 ± 1.05 29.7 ± 2.3 37.5 ± 3.3 8.9 ± 1.3 CO 122 ± 12 69 ± 4 3± 1 58 ± 4 8.08 ± 0.97 35.4 ± 2.2 40.1 ± 2.7 6.7 ± 1.4 C3 110 ± 10 70 ±4 3 ± 1 63 ± 3 7.32 ± 0.66 29.4 ± 2.9 35.5 ± 4.6 8.0 ± 2.0 SCS-CO 128 ±13 70 ± 5 2 ± 1 58 ± 3 8.62 ± 0.89 35.3 ± 2.5 39.8 ± 2.8 6.9 ± 1.4Recovery 113 ± 11 74 ± 4 1 ± 1 67 ± 3 8.22 ± 1.01 29.9 ± 2.1 35.6 ± 2.98.2 ± 1.3

[0333] The decrease in LV chamber systolic and diastolic pressures wasnot significant presumably due to the short duration of the individualischemic periods. Although the heart rate/LV pressure product, an indexof myocardial oxygen demand, increased slightly during acute ischemia,this index did not change significantly due to large standard deviationsfrom mean values data were not normalized. Ventricular dynamics were notsignificantly altered with acute LAD occlusion and concurrent SCS.Cardiac hemodynamics at the end of the experimental protocol i.e.,recovery—10 min after the final intervention was comparable to baselinevalues.

[0334] Regional Myocardial Blood Flow Distribution

[0335] The overall anatomic risk zone represented 21.2 “5.3% mean”1 S.D.of total LV volume. Distribution of ventricular blood flow determined byradiolabeled microspheres showed that average blood flow levelsdecreased significantly within the ischemic zone during LAD occlusion0.9“0.1 to 0.2”0.1 ml/min/g p-0.02. Blood flow levels were notsignificantly affected in the non-ischemic LV wall, the right ventricleor kidneys during LAD occlusion Table IV.

[0336] Table IV Summary of Blood Flow Changes

[0337] Data are means ±S.E.M. Data are expressed in ml/min/g wet weight.Abbreviatons are indicated in Table III. C1 SCS C2 CO C3 SCS-CO Ischemiczone Endocardium  1.00 ± 0.016 1.02 ± 0.08 1.02 ± 0.27 0.24 ± 0.06 1.53± 0.43 0.28 ± 0.09 Mid- 0.80 ± 0.15 0.76 ± 0.12 0.84 ± 0.25 0.24 ± 0.061.29 ± 0.29 0.29 ± 0.06 myocardium Epicardium 0.82 ± 0.18 0.75 ± 0.100.90 ± 0.20 0.28 ± 0.08 1.10 ± 0.27 0.24 ± 0.06 Non-ischemic ZoneEndocardium 1.07 ± 1.12 1.07 ± 0.1  1.14 ± 0.25 1.05 ± 0.20 1.67 ± 0.391.04 ± 0.17 Mid- 0.91 ± 0.11 0.95 ± 0.11 1.10 ± 0.14 0.93 ± 0.18 1.32 ±0.36 0.94 ± 0.19 myocardium Epicardium 0.72 ± 0.09 0.84 ± 0.13 0.84 ±0.13 0.82 ± 0.18 1.05 ± 0.29 0.77 ± 0.13 Right 0.54 ± 0.07 0.856 ± 0.08 0.70 ± 0.12 0.78 ± 0.14 0.78 ± 0.14 0.44 ± 0.07 ventricle Kidney Inner0.29 ± 0.03 0.31 ± 0.04 0.30 ± 0.06 0.49 ± 0.10 0.49 ± 0.10 0.60 ± 0.07(medulla) Outer 3.46 ± 0.36 3.49 ± 0.23 4.60 ± 1.05 4.88 ± 1.15 4.88 ±1.15 3.24 ± 0.42 (cortex)

[0338] During application of SCS concomitant with LAD occlusion, thelevel of blood flow reduction in the ischemic zone was similar to thatwhich occurred during LAD occlusion alone Table IV and FIG. 37 SCS didnot affect transmural blood flow distribution within the LV-free wall orthe intraventricular septum (FIG. 37), or total ventricular flows.Neither did SCS affect regional blood flow in the kidneys Table IV.

[0339] Pressure-Volume Relations

[0340] The LV pressure-volume loops did not change during SCS FIGS.38(A) and (B). The LV pressure-volume loops changed immediately afterthe onset of LAD occlusion. The LV volumes shifted rightward, whilesimilar peak FIG. 37. Transmural blood flow ml/min/g to LV ischemicclosed spheres and non-ischemic closed squares zones for each of thethree baseline control conditions C1, C2 and C3 and during thesuccessive interventions of 5-min spinal cord stimulation SCS, 4-minocclusion of the LAD CO , and concurrent 5-min SCS plus 4 min LADocclusion commencing 1 min into SCS SCS-CO. Transmural blood flow withinthe ischemic zone is significantly lower ps0.02 during both CO, andSCS-CO psNS between these two interventions compared to baselinesystolic pressures were generated FIG. 38(C) and (D). LV stroke volumeand ejection fraction was reduced almost 30% compared to baseline valuesduring the periods of local ventricular ischemia. Corresponding changesin LV pressure-volume loops were observed when the LAD was occludedconcurrent with SCS FIG. 38(E) and (F). LV stroke volume and ejectionfraction were similarly diminished. Thus, SCS did not improve overallventricular dynamics in the presence of local myocardial ischemia.

DISCUSSION

[0341] Neuromodulation therapy is utilized to alleviate angina ofcardiac origin. In order to investigate the underlying mechanisms forsuch therapy, we studied the potential influence of SCS on myocardialblood flow and LV dynamics in the normal canine heart. The results ofthis study demonstrate that electrical stimulation of the upper thoracicspinal cord does not alter either transmural distribution of blood flowwithin the myocardial wall or overall LV dynamics. As has been found inthe past (Foreman et. al., 2000), transient focal myocardial ischemiadid not alter left-ventricular chamber systolic or diastolic pressuressignificantly Table III. On the other hand, the LV pressure-volume loopwas shifted rightward during transient occlusion of the LAD indicativeof volume changes FIG. 38. Changes in LV volumes were accompanied by asignificant decline in the LV ejection fraction, synonymous with alteredcontractile function elicited by acute coronary artery occlusion (Pauluset al., 1985; Sasayama et al., 1985; Applegate, 1991). As expected,transmural blood flow was also reduced in the ischemic zone. Applicationof SCS during this ischemic challenge did not further alter regionalmyocardial blood flow. Neither did SCS affect the rightward shift of LVpressure-volume loops induced during the ischemic challenge.

[0342] Limitations of Study

[0343] The radioactive microsphere technique for determination ofregional blood flow distribution has the advantage that microspheres aretrapped during the first pass through an organ with no detectablerecirculation; however, the number of microspheres that can be safelyinjected without affecting cardiac hemodynamics is finite. Baer et al.(1984) estimated that injection of 18-27 million microspheres ninedifferent radiolabels of 2-3 million spheres each into the left atriumhad little influence on distribution of blood flow during normalcoronary autoregulation or vasodilatation. In the present study systolicLV pressure and mean aortic pressure remained constant during and aftermicrosphere injections; this indicates that these dogs werehemodynamically stable during the respective experimental protocols. Itis known that radioactive microspheres have the inherent limitation thatregional blood flow changes less than 10% of baseline are not readilydetectable. Thus, minor changes in regional blood flow distributionsbetween myocardial regions might not be detected. Regardless, the lackof change in measured cardiac indices during SCS suggests that theprimary determinant of regional myocardial blood flow and cardiac workwas not altered thereby. Coronary vascular resistance or conductance wasnot calculated in the present study since we did not includemeasurements of extravascular compressive forces or critical closingpressure; in a recent study from our laboratory we document that duringautoregulation the entire coronary pressure-flow relation can shift inrelation to changes in LV pressure and volume Rouleau et. al., 1999 .Under steady-state conditions in the present study theendocardialrepicardial blood flow ratio was similar not during ischemia;as such, distribution of blood flow was maintained across the LV wall.

[0344] Major differences exist between the present study and someclinical studies; we used SCS, while Chauhan et al. (1994), who reportedan increase in blood flow in the contralateral coronary artery usedTENS. In addition, SCS at 90% motor threshold in the anesthetized caninerepresents an intensity used clinically when patients anticipate morestrenuous activities. Normally, stimulation intensities between 60% and66% of paresthesia threshold are adequate to reduce anginal pain.

[0345] Clinical Implications of SCS

[0346] Pain-reducing properties of neuromodulation resulting from SCSare based on the gate theory of pain (Melzack and Wall, 1965). Thistheory proposes that stimulation of large afferent fibers conductinginnocuous information reduce the nociceptive effects of the smallafferent fibers on the activity of spinal neurons. Neuromodulation isknown to stimulate neurons in the dorsal horn (Melzack and Wall, 1965;Chandler et al., 1993) and higher centers (Hautvast et al., 1997;Yakhnitsa et al., 1999). Recently we documented that the activitygenerated by intrinsic cardiac neurons is also suppressed by SCS, evenduring acute myocardial ischemic challenges (Foreman et al., 2000).

[0347] The present results are in agreement with the majority ofprevious clinical studies that indicate a lack of effect of SCS onoverall coronary blood flow (De Landesheere et al., 1992; Hautvast etal., 1996; Sanderson et al., 1996; Norrsell et al., 1998). SCS wasapplied for 5 min in our study, while in most clinical studies it ismaintained for much longer time periods. It is unlikely that theduration of SCS stimulation determines the effects that thisintervention exerts on coronary artery blood flow (Chauhan et al.,1994). The present study was performed in canine hearts that underwentbrief periods of regional ventricular ischemia. In clinical studiescarried out among patients with stable angina, electrical orpharmacological i.e., dipyramidole induction of cardiac stress in thepresence of neuromodulation has been shown to exert no influence ontheir coronary blood flow (Hautvast et al., 1996; Norrsell et al.,1998). In the present study, the canine coronary vasculature wasconsidered to be normal in contrast to these clinical investigations inwhich coronary artery blood flow was assessed in patients withunderlying coronary vessel disease. The primary determinant of bloodflow in the normal myocardium is regional myocardial metabolic demand,the latter being very dependent on LV dynamics (Hoffman, 1987).Hemodynamic alterations are accompanied by changes in distribution ofblood flow patterns across the LV wall (Dole and Bishop, 1982; Messinaet al., 1985). For that reason, it is important to note that the periodsof regional ventricular ischemia induced in these experiments were ofshort enough duration to induce minor, if any change in left-ventricularpressure Table III. It is also important to point out that thehemodynamic results obtained in the canine model may not directly applyto other animal models with different coronary collateral vascularfunction. However, intrinsic cardiac neuronal results derived from thecanine model appear to be applicable to the porcine model and even tohumans undergoing bypass surgery. Thus, the effects of regionalventricular ischemia on the intrinsic cardiac nervous system depend moreon the location of the neurons and the site of ventricular injury thanon species investigated. The ischemic area i.e., anatomic risk zone thatwas produced in these experiments was significant, being 21.2 “5.3%mean” S.D. of the total ventricular volume.

[0348] Ventricular ischemic zones of this magnitude are sufficient toinduce fatal ventricular arrhythmias (Vegh et al., 1991; Curtis et al.,1989). In the study reported by Vegh et al. (1991), hearts werepreconditioned by repeated episodes of rapid ventricular pacing; thisresulted in significant cardioprotection against ischemia-inducedventricular arrhythmias. Whether SCS triggered a preconditioningresponse in the present experimental model is debatable. The lack ofheart rate or hemodynamic effect, reflected by the similarity of themyocardial oxygen demand and myocardial blood flow data, indicates thatSCS may not have induced a preconditioning response. In addition, we didnot observe an increase in coronary collateral flow within the ischemicmyocardium. Whether preconditioning increases coronary collateral bloodflow within the ischemic zone in dogs remains unclear. In the presentstudy, ischemic zone size was not influenced by SCS. These data are inaccord with the fact that SCS did not affect the LV pressure-volumerelationships in addition to arterial perfusion of ventricular tissue.We cannot completely exclude the possibility that SCS redistributesblood flow between adjacent myocardial regions via the coronarycollateral circulation since the microsphere technique may not reliablydetect changes in blood flow at this level. Rather, these data suggestthat the anti-anginal effects of SCS are induced by mechanisms otherthan changes in regional myocardial blood flow or

LV DYNAMICS

[0349] Summary and Conclusions

[0350] Data obtained in the present study document the fact that SCSdoes not affect either total myocardial blood flow or blood flowdistribution across the LV wall. Neither does SCS affect thedistribution of blood within the ischemic myocardium, nor that betweenischemic and non-ischemic zones.

[0351] Additional studies hereinafter disclosed herein, demonstrate that(1) ischemia causes neuronal activation; (2) SCS or DCA stimulationnullifies or quenches such ischemia induced activated neurons; and (3)nullification and/or such quenching or suppressor effects on suchactivated neurons occur (i) prior, (ii) during, and (iii) after thecessation of SCS or DCA stimulation. Thus, SCS or DCA effectivelydirects the intrinsic nervous system in such a manner as to have animmediate and lasting effect on the activity of myocardial neurons andthe intrinsic cardiac nervous system in general.

[0352] As mentioned previously, it is known currently that electricalexcitation of the dorsal aspect of the rostral thoracic spinal cordimparts long-term therapeutic benefits to patients with angina pectoris.What is not known and is being claimed and disclosed in the presentapplication, is that spinal cord stimulation induces short-termsuppressor effects on the intrinsic cardiac nervous system. The resultsof the following tests show that spinal cord stimulation (SCS) induceslong-term effects on the intrinsic nervous system, particularly in thepresence of myocardial ischemia.

[0353] The activity generated by right atrial neurons was recorded in 10anesthetized dogs during basal states, during prolonged (15 min)occlusion of the left anterior descending coronary artery, and duringthe subsequent reperfusion phase. Neuronal activity and cardiovascularindices were also monitored when the dorsal T1-T4 segments of the spinalcord were stimulated electrically (50 Hz; 0.2 ms) at an intensity 90% ofmotor threshold (mean 0.32 mA) for 17 min. SCS was performed before,during and after 15-min periods of regional ventricular ischemia.Occlusion of a major coronary artery, one that did not perfuseinvestigated neurons, resulted in their excitation. Ischemia-inducedneuronal excitatory effects were suppressed (>76% from baseline) by SCS.SCS suppression of intrinsic cardiac neuronal activity persisted duringthe subsequent reperfusion period; after terminating 17 min of SCS, atleast 20 min elapsed before intrinsic cardiac neuronal activity returnedto baseline values. It is concluded that populations of intrinsiccardiac neurons are activated by inputs arising from the ischemicmyocardium. Ischemia-induced activation of these neurons is nullified bySCS. The neuronal suppressor effects that SCS induces persist not onlyduring reperfusion, but also for an extended period of time thereafter.

INTRODUCTION

[0354] High frequency, low intensity electrical stimulation of thedorsal aspect of the T1-T2 spinal cord alleviates angina pectoris inpatients suffering from ischaemic heart disease (Eliasson et al., 1996;Mannheimer et al., 1993; Sanderson et al., 1992). The therapeuticeffects of spinal cord stimulation on angina (SCS) can persist for hoursafter its termination (Jessurun et al., 1999). Accumulating evidencedemonstrates that SCS is a safe anti-anginal treatment modality thatdoes not result in increased frequency of arrhythmia formation(DeJongste et al., 1994; Eliasson et al., 1996; Hautvast et al., 1998;Mannheimer et al., 1998). However, the mechanisms whereby SCS producesits long-term effects remain unknown. Clinical studies have led to thehypothesis that SCS exerts its anti-anginal effects principally byaltering the ventricular oxygen supply/demand ratio (Mannheimer et al.,1993; Sanderson et al., 1992). Mannheimer et al. (1993) suggested thatSCS reduces cardiac metabolism, thereby reducing oxygen demand and,consequently, the myocardial lactate production within the ischemicmyocardium. In this regard, Hautvastet al. (1998) proposed that SCSredistributes myocardial blood flow from normal to ischaemic regions ofthe heart. However, in the canine model, SCS does not alter cardiacchronotropism or inotropism (as shown in the experiments above),suggesting that oxygen demand is minimally affected by such anintervention. Furthermore, SCS does not alter blood flow distributionwithin either the normal or ischaemic canine myocardium (also shownabove).

[0355] As we show, the effects of SCS reflect changes within the CNSand/or changes in neurohumoral control of the heart. SCS modulatesimpulse transmission within the spinothalamic tracts of the spinal cordwithout blocking afferent neuronal signals arising from the ischaemicmyocardium (Chandler et al., 1993). It also alters intrinsic cardiacneuronal function (show hereinabove). The intrinsic cardiac nervoussystem represents the final common regulator of regional cardiacfunction (Armour, 1991; Ardell, 2000). Its neurons are under theconstant influence of central neurons, including those in the spinalcord (Gagliardi et al., 1988). Regional myocardial ischaemia results inthe heterogeneous activation of the intrinsic cardiac nervous system(Armour et al., 1998). When sub-populations of intrinsic cardiac neuronsbecome excessively activated, the cardiac electrophysiologicalconsequences, such as the occurrence of ventricular tachycardia orventricular fibrillation, may be devastating (Armour, 1991).Stabilization of the intrathoracic intrinsic cardiac nervous system,especially in the presence of myocardial ischaemia ameliorate thepotential for cardiac electrical instability. Such a system is shown anddemonstrated in the experiments outlined in the present application.

[0356] Short duration SCS (4 min) transiently suppresses the activitygenerated by intrinsic cardiac neurons (shown hereinabove). In aclinical setting, the anti-anginal effects of SCS persist long after itstermination (Jessurun et al., 1999). The following experiments weredevised to evaluate the effects of prolonged (17 min) SCS on theintrinsic cardiac nervous system in normally perfused and ischaemichearts. These experiments were also designed to evaluate whether theneurohormonal effects that SCS imparts on the intrinsic cardiac nervoussystem persist not only throughout its application, but also for a timethereafter.

MATERIALS AND METHODS

[0357] Animal Preparation

[0358] The Institutional Animal Care and Use Committee of DalhousieUniversity approved the experiments performed in the followingexperiments. These experiments followed the guidelines outlined by theInternational Association for the Study of Pain as well as the NIH Guidefor the Care and Use of Laboratory Animals (National Academy Press,Washington, DC, 1996). Ten adult dogs of mixed breed, weighing between12.5 and 26 kg (mean 19.6 kg), were used for this study. The animalswere kept under standard laboratory conditions in a light-cycledenvironment (12 h/12 h) with free access to water at all times and tofood at regular intervals.

[0359] Dogs were anesthetized in a standard manner by firstadministering a bolus dose of sodium thiopental (20 mg kg⁻¹, i.v.).Anesthesia was maintained throughout the surgery period by means ofbolus doses of thiopental (5 mg kg⁻¹, i.v.) administered to effect every5-10 min. Animals were intubated and then artificially ventilated usinga Bird Mark VII respirator with 100% O₂. After completing the surgery,anesthesia was changed to alpha chloralose by first administering a doseof alpha chloralose (75 mg kg⁻¹, i.v.). Thereafter, repeat doses ofalpha chloralose (20 mg kg⁻¹, i.v.) were administered, as required,during the remainder of the experiments.

[0360] The level of anesthesia was checked throughout each experiment byobserving pupil reaction as well as monitoring jaw tension, heart rateand blood pressure, and by periodically checking for the withdrawalreflex by squeezing a paw. Since each bolus of alpha chloralosesuppressed neuronal activity for a few minutes after its administration,these doses were administered between the interventions performed ineach protocol. This anesthetic regimen produces adequate anesthesiawithout inordinately suppressing peripheral autonomic neural activity.Electrodes were inserted in the forelimbs and the left hind limb andconnected to an Astro-Med (West Warwick, R.I.) model MT 9500eight-channel rectilinear recorder to monitor a Lead IIelectrocardiogram throughout the experiments. In addition, a 12-leadelectrocardiogram (ECG) strip-chart recorder (Nihon Ohden Cardiofax Vmodel BME 7707) was employed to obtain standard lead electrocardiogramsduring control states and at 5-min intervals during each intervention.Heart rate and the duration of the PQ, QR and QTc intervals wereanalyzed during control states as well as 1, 5, 10 and 15 min afterocclusions began in the absence or presence of SCS. In addition,alterations in the morphology of ST-T segments and arrhythmia formationwere assessed.

[0361] Implantation of Spinal Cord Stimulation Electrodes

[0362] After induction of anesthesia, animals were placed in the proneposition. The epidural space of the mid-thoracic spinal column waspenetrated percutaneously with a Toughy needle (15 F). A Toughy needlehas a slight angle at its tip to ease penetration between vertebralprocesses. Using the loss-of-resistance technique as is routinely donein a clinical setting, the tip of the Toughy needle was slowly advanceduntil it entered the epidural space, as visualized via A-P fluoroscopy.Once the inner cannula was removed from the Toughy needle, a four-polecatheter electrode (Medtronic QUAD Plus Model 3888; Medtronic,Minneapolis, Minn.) was introduced through the needle such that its tipcould be advanced to the T1 level of the spinal column, as determined byfluoroscopy. The tip of this electrode was positioned slightly to theleft of the midline, as is done in a clinical setting (Linderoth andForeman, 1999). The rostral and caudal poles of the stimulatingelectrode chosen for subsequent use (inter-electrode distance of 1.5 cm)were located at the levels of the T1 and T4 vertebrae. Correct placementof the stimulating electrodes was confirmed by delivering electricalcurrent to induce motor responses using the rostral or caudal poles ascathodes, respectively.

[0363] The rostral cathode (T1 level) and caudal anode (T4 level) of thequadripolar electrode were connected to a Grass S88 stimulator via aconstant current stimulus isolation unit (Grass model CCU1 and GrassSIU5). Stimuli, delivered at 50 Hz and 0.2-ms duration, were monitoredon an oscilloscope to determine the amount of current delivered. Rostralstimulation above motor threshold resulted in proximal forepaw orshoulder muscle fasciculations (or both), while caudal electrodestimulation induced contractions in the thoracic trunk. When theappropriate electrode position was confirmed, the electrode lead wascovered by a Teflon protective sleeve and fixed to adjacent interspinousligaments with a suture. Extension wires attached to the electrode leadswere connected to the Grass constant current stimulator (seehereinabove). Motor responses were rechecked after the animal had beenplaced in the supine position to ensure that the electrodes had notmoved during that maneuver.

[0364] Cardiac Instrumentation

[0365] After placing the animal on its back, a bilateral thoracotomy wasmade in the fifth intercostal space. The ventral pericardium was incisedand retracted laterally to expose the heart and the ventral right atrialdeposit of fat containing the ventral component of the right atrialganglionated plexus. We investigated the activity generated by neuronsin the right atrial ganglionated plexus because not only are theyrepresentative of those found in other atrial and in ventricularganglionated plexuses (Armour, 1991), but they do not receive theirarterial blood supply from the left ventral descending coronary artery(Huang et al., 1993). The regional arterial blood supply of theseneurons and other cardiac tissues is unaffected by spinal cordstimulation (Kingma et al., in press). Thus, the blood supply ofidentified neurons was not affected in a significant manner by theprocedures described below.

[0366] Left ventricular chamber pressure was monitored via a Cordis(Miami, Fla.) #7 French pigtail catheter that was inserted into thechamber via one femoral artery. Systemic arterial pressure was measuredusing a Cordis #6 French catheter placed in the descending aorta via theother femoral artery. These catheters were attached to Bentley (Irvine,Calif.) Trantec model 800 transducers.

[0367] Neuronal Recording

[0368] To minimize epicardial motion during each cardiac beat, acircular ring of stiff wire was placed gently on the fatty epicardialtissue overlying the ventral surface of the right atrium containing theright atrial ganglionated plexus (Gagliardi et al., 1988). A tungstenmicroelectrode (10-mm shank diameter; exposed tip of 1 mm; impedance of9-11 MV at 1000 Hz) mounted on a micromanipulator was lowered into thisfat using a microdrive. The indifferent electrode was attached tomediastinal connective tissue adjacent to the heart. The electrode tipexplored this tissue at depths ranging from the surface of the fat toregions adjacent to cardiac musculature. Proximity to the atrialmusculature was indicated by increases in the amplitude of the ECGartifact. Signals generated by the somata and/or proximal dendrites ofintrinsic atrial neurons were differentially amplified by a PrincetonApplied Research model 113 amplifier with bandpass filters set at 300 Hzto 10 kHz and an amplification range of 100-500×. The output of thisamplifier, further amplified (50-200×) and filtered (bandwidth 100 Hz-2kHz) by means of optically isolated amplifiers (Applied MicroelectronicsInstitute, Halifax, NS, Canada), was led to a Nicolet model 207oscilloscope and to a Grass AM8 Audio Monitor. Signals were displayed onan Astro-Med MT 9500 eight-channel rectilinear recorder along with thecardiovascular variables described above. All data were stored via aVetter (Rebesburg, Pa.) M3000A digital tape system for later analysis.Action potentials generated by neurons in a site in the right atrialganglionated plexus were recorded,

[0369] Individual units being identified by their amplitudes andconfigurations. The amplitudes of the identified action potentialsvaried by less than 10-50 μV over several hours; individual actionpotential retained the same configuration over time. Individual actionpotentials so identified are generated by somata and/or dendrites ratherthan axons of passage. Action potentials recorded at; a given locus thatdisplayed the same configuration and amplitude were considered to begenerated by a single unit. When multiple action potentials wereidentified at an active site, action potentials generate by individualunits were discriminated by means of a window discriminator (HartleyInstrumentation Development Laboratories, Baylor College of Medicine,Houston, Tex.).

[0370] Induction of Coronary Artery Occlusion

[0371] A silk (3-0) ligature was placed around the left anteriordescending (LAD) coronary artery approximately 1.5 cm from its origin,distal to its first diagonal branch. If a relatively large number ofcollateral arterial branches from the apex or lateral wall were evident,ligatures were also placed around these vessels. These ligatures wereled through short segments of polyethylene tubing in order to occludethese arteries later in the experiments. Since the arterial blood supplyof investigated right atrial neurons arises from major branches of theright and distal circumflex coronary arteries, their blood supplyremained patent during these coronary artery occlusions.

[0372] Spinal Cord Stimulation (SCS)

[0373] With the animal placed in the supine position, the intensity ofthe current delivered via the bipolar electrode was increased until adetectable skeletal muscle motor response was evident, as describedabove. This current intensity corresponds to the threshold for motoractivity induction (MT). An intensity of 90% of MT was used for allsubsequent stimuli as it recruits A-delta fibers and other axonpopulations (Linderoth and Foreman, 1999). This stimulus intensitycorresponds to parameters used clinically to stimulate the thoracicspinal cord (Linderoth and Foreman, 1999). The stimulus intensity at 90%MT varied between 0.09 and 0.63 mA (mean 0.32 mA) among animals studied.Presumably the variation in current intensity at 90% MT among animalsreflected slight differences in electrode position with respect to thedorsal surface of the thoracic cord. The MT was checked periodically andfound to remain constant over time in individual animals.

PROTOCOLS

[0374] Two separate protocols were applied to each of five animals, theorder of their application being randomized among the 10 animals. Thesewere devised to evaluate the long-term effects of successive 15-minperiods of coronary artery occlusion performed with or withoutconcurrent SCS. Electrical stimuli were delivered to the dorsal aspectof the thoracic spinal cord for 17-min periods. Protocol #1 began withtwo 15-min periods of coronary artery occlusion, with a 1.5-h intervalelapsing between occlusions (FIG. 39, top panels). The coronary arteryocclusion was repeated in these five animals in order to determine thereproducibility of ischaemia-induced changes in ECG morphology andintrinsic cardiac neuronal activity. After an additional 1.5-h recoveryphase, 17 min of SCS (90% MT) was performed during which time a 15-minperiod of coronary artery occlusion was instigated 1 min after SCSbegan. This was followed by a 1-h period during which time neuronalactivity was quantified. Thereafter, veratridine was applied toepicardial loci (see hereinbelow).

[0375] Protocol #2 was employed in the other five animals. In protocol#2, the effects of 17 min of SCS combined with 15 min of coronary arteryocclusion were studied first. The coronary occlusion was initiated 1 minafter beginning SCS (FIG. 34, bottom panels). After waiting for 1.5 h, a15-min period of coronary artery occlusion was performed alone. Afterwaiting another 1.5 h, the combined SCS and coronary artery occlusionwas performed again. Protocol #2 was performed to verify thereproducibility of effects induced by SCS in the presence of ventricularischaemia. This protocol was followed by a 1-h recovery period afterwhich time veratridine was applied to epicardial loci.

[0376] Epicardial Application of Veratridine

[0377] Veratridine is a selective modifier of Na+ channels that excitessensory neurites associated with cardiac afferent neurons withoutinducing tachyphylaxis (Thompson et al., 2000). This agent (obtainedfrom Sigma, St. Louis, Mo., USA) was dissolved in physiological Tyrodesolution to make a 7.5 μM solution. Gauze squares (1×1 cm) soaked withveratridine (0.5 ml) were applied for 60-100 s to discrete epicardialloci on the right ventricular conus and the ventral surface of the leftventricle at the end of each experiment (n=10 dogs). In four animals,the effects that epicardial application of veratridine exerted on theintrinsic cardiac nervous system were also tested before the protocolsdescribed above had been performed. After removing the applied gauze,the epicardial region was flushed with normal saline for at least 30 s.Gauze squares soaked with room temperature normal saline were alsoapplied to identify epicardial sensory fields in order to determinewhether neuronal responses elicited by chemical application were due tovehicle effects or the mechanical effects elicited by gauze squares.

DATA ANALYSIS

[0378] Individual action potentials generated by the somata or dendritesof neurons within the right atrial ganglionated plexus were averagedover 30-s periods of time prior to and during each intervention. Averageheart rate, left ventricular chamber systolic pressure and aorticpressure were determined concomitantly. Changes in ECG morphologyinduced by the protocols were assessed. When the coronary arteryocclusion was performed alone, data were assessed during baselineconditions and 14 min after the occlusion began (occlusion period), aswell as starting 15 s after reperfusion began (reperfusion period). Whenthe occlusions were performed in the presence of SCS, cardiac indicesand neuronal activity were assessed at five time points: (1) controlperiod; (2) 30 s after SCS began; (3) 12 min after coronary arteryocclusion began, in the presence of SCS; (4) after terminating theocclusion while the SCS persisted; and (5) within 30-60 s of terminatingthe SCS. Data are expressed as means ±S.E.M. One-way ANOVA and pairedt-test, with Bonferroni correction for multiple tests, were employed toexamine grouped responses elicited during occlusion of a coronary arteryalone (first occlusion) or when SCS and occlusions were performed ineach protocol. Values of P<0.05 were used to Determine Significance

RESULTS

[0379] Identification of Active Sites

[0380] Action potentials with signal-to-noise ratios greater than 3:1were identified in 2-3 loci within the ventral right atrial ganglionatedplexus of each animal. Based on the different amplitudes andconfigurations of action potentials recorded at one site per animal, anaverage of 3.2±0.5 (range 2-6) neurons generated spontaneous activity atinvestigated sites during control states. Neuronal activity during basalstates was usually sporadic in nature. During basal states, a fewspontaneous active neurons were identified in active loci of mostanimals (FIG. 40), while in a few animals, a number of neurons generatedspontaneous activity (FIG. 41(D)). The neuron aggregates identified inone active locus in each of the 10 investigated dogs generated, onaverage, 34.1±3.4 to 48.2±6.5 impulses/min (Table V). TABLE VIntervention HR LVP Neuronal activity (n = 10 dogs) (bpm) (mmHg) AP(mmHg) (impulses/min) Control 134 ± 2 134± 5 138 ± 5/99 ± 5 34.1 ≠ 3.4CAO 134 ± 2 136 ± 5 140 ± 5/99 ± 5  62.2 ≠ 9.5* Reperfusion 134 ± 2 136± 5 138 ± 5/99 ± 5  66.0 ≠ 13.3* Control 130 ± 3 137 ± 4 141 ± 5/99 ± 548.2 ≠ 6.5 SCS 130 ± 3 137 ± 4 141 ± 5/99 ± 6  15.1 ≠ 3.1* SCS + CAO 128± 3 139 ± 4 141 ± 5/98 ± 6  13.5 ≠ 2.4* SCS + 130± 3 137 ± 4 141 ± 5/99± 5  15.2 ≠ 3.3* reperfusion Control 131 ± 4 134 ± 5 141 ± 5/99 ± 5 46.8 ≠ 10.2

[0381] Effects of Transient Myocardial Ischaemia

[0382] Monitored cardiac indices did not change significantly overallduring coronary artery occlusion or the reperfusion period, except whencardiac arrhythmias occurred. For instance, heart rate was 134±2beats/min (bpm) before occlusion and 130±3, 134±3, 132±2 and 134±2 bpmafter 1, 5, 10 and 15 min of ischaemia, respectively. S-T segmentalterations and terminal QRS slurring was evident in the ECG pattern ofeach animal during ischaemic episodes (FIG. 42). The ST segmentsremained altered (elevated or depressed by 1.0±0.2 mm) during the first2-5 min of reperfusion. ECG patterns returned to baseline values within20 min of reestablishing coronary artery blood flow. Short bursts ofventricular arrhythmias occurred in most animals during coronary arteryocclusion. In two animals, ventricular fibrillation developed during orimmediately after the first coronary artery occlusion. In thoseinstances, the hearts were successfully defibrillated and, after 1 h,the protocol was continued. These animals did not exhibit any unusualalterations in monitored indices throughout during the rest of theprotocols. The data obtained during these short bouts of arrhythmias orfibrillation were excluded from the study.

[0383] Overall, these electrophysiological data substantiate thesubstantial ischaemia insult that was induced by 15-min periods of leftanterior descending coronary artery (LAD) occlusion. When the LAD wasoccluded in either protocol in the absence of SCS, the activitygenerated by right atrial neurons (FIG. 40). Effects of coronary arteryocclusion on the activity generated by intrinsic cardiac neurons in oneanimal. Following occlusion of the left anterior descending coronaryartery (beginning at arrow below), the activity generated by rightatrial neurons (lowest line) increased (right-hand panel). Heart ratewas unaffected by this intervention, while left ventricular chambersystolic pressure (LVP) increased a little. The time between panelsrepresents 1.5 min. increased by 82% (FIG. 40; Table V). Neuronalexcitation persisted through the period of occlusion. During protocol#1, the two successive 15-min periods of coronary artery occlusionseparated by 1.5 h of recovery induced similar neuronal excitation.Twelve minutes after initiating the first LAD occlusion, neuronalactivity was 69% greater than identified in normally perfused states(31.7±6.9 to 53.5±10.2 impulses/min; P<0.01). During the second periodof coronary artery occlusion, neuronal activity increased by 95%(28.3±4.1 to 55.1±8.9 impulses/min; P<0.01). Neuronal activity began toincrease within 30-45 s after coronary artery occlusion began. Thisoccurred despite the fact that coronary artery occlusion did notinterfere with the arterial blood supply to identified right atrialneurons as it arose from the right and distal circumflex coronaryarteries. Furthermore, neuronal activity remained elevated not onlythroughout the period of occlusion but during the early reperfusionperiod following reestablishing coronary artery flow. Five to tenminutes after reestablishment of coronary artery flow, neuronal activitybegan to diminish, reaching steady state values within 15 min.

[0384]FIG. 41 shows the activity generated by intrinsic cardiac neuronsin one animal during control states (panel A, lowest line) decreasedwhen the dorsal aspect of the spinal cord was stimulated (panel B). Thesuppressor effects of SCS persisted during coronary artery occlusion(panel C). The electrical stimuli delivered during SCS are representedin panels B and C by regular, low signal-to-noise artifacts (note thatatrial electrical artifact is recorded during each cardiac cycle as alow signal during the p wave of the ECG). The suppression of spontaneousactivity generated by intrinsic cardiac neurons persisted afterdiscontinuing SCS (panel E represents neuronal activity recorded 5 minpost-SCS and 6 min post-LAD occlusion; panel D represents basal activityat same time scale obtained before commencing these interventions).ECG=electrocardiogram; AP=aortic pressure; LVP=left ventricular chamberpressure.

[0385] Table V shows heart rate (HR), left ventricular chamber systolicpressure (LVP), aortic systolic and diastolic pressures (AP) and theactivity generated by right atrial neurons recorded before (control) andduring coronary artery occlusion (CAO), as well as during earlyreperfusion (reperfusion). These indices were also recorded whenocclusion occurred during spinal cord stimulation.

[0386] Effects of Spinal Cord Stimulation in the Presence of MyocardialIschaemia

[0387] During normal coronary artery perfusion, SCS did not alter theECG or monitored cardiac indices (Table V). The activity generated byidentified right atrial neurons was reduced from 48.2±6.5 to 23±2.5impulses/min within 30 s of applying electrical current to the dorsalaspect of the rostral thoracic spinal cord in hearts with normalcoronary arterial blood supply (FIG. 43). After the coronary arteryocclusion had been maintained for 1 min in the presence of SCS (2 minafter beginning SCS), right atrial neuronal activity was reduced to15.1±3.1 impulses/min. Thus, SCS suppressed the activity generated byintrinsic cardiac neurons not only in normally perfused hearts (FIG.41(B)), but also in the presence of regional ventricular ischaemia (FIG.41(C)). Furthermore, the neuronal suppressor effects of SCS persistedthroughout the ischaemic periods. Monitored cardiovascular indices didnot change overall when SCS was applied during coronary arteryocclusion. Ischaemia-induced alterations in ECG patterns also remainedthroughout the period when SCS was applied concomitant with theocclusions. Neuronal activity gradually increased after discontinuingSCS such that by 20 to 25 min after terminating SCS, neuronal activitywas similar statistically to that recorded during basal conditions. Itincreased a little thereafter (FIG. 43).

[0388] Epicardial Application of Veratridine

[0389] The activity generated by right atrial neurons increased whenveratridine was applied topically to their ventricular sensory inputs.Veratridine-induced excitation of intrinsic cardiac neurons was studiedboth before and after application of SCS in four animals. In thosecases, veratridine enhanced intrinsic cardiac neuronal activity by 124%(40.5±26.7; P<0.05) before application of SCS and by only 39% (11.8±2.5to 16.5±4.6 impulses/min; no significant difference) after itsapplication. When veratridine was applied to their ventricular sensoryinputs after completing the protocols in all 10 dogs (following SCS andregional ventricular ischaemia), intrinsic cardiac neuronal activityincreased by only 58% (25.6±5.7 to 40.6±12.5 impulses/min; P<0.05).

[0390] FIG. II shows representative ECG records obtained from one animalduring control states (A), as well as a few minutes after beginningcoronary artery occlusion in the presence of spinal cord stimulation (B)and at the end of occlusion while SCS was maintained (C). Note that STsegment alterations occurred throughout the period of ischaemia.

Discussion

[0391] The results obtained from the experiments conducted in thepresent study not only confirm that spinal cord neurons can modulate theintrinsic cardiac nervous system (as discussed hereinabove), but theydemonstrate that such modulation persists unabated throughout 17-minperiods of stimulating the dorsal thoracic spinal cord. They alsoindicate that spinal cord neurons continue to exert their suppressoreffects on the intrinsic cardiac nervous system long after theiractivation terminates. Furthermore, these data indicate that spinal cordneurons reorganize information processing within the intrinsic cardiacnervous system arising from the ischaemic myocardium, including duringthe reperfusion post-ischaemic phase. Finally, as indicated by theneural responses evoked by veratridine application to the ventricularepicardium, the stabilizing influence that SCS exerts on the intrinsiccardiac nervous system extends to intrinsic cardiac reflex responsesevoked by activating cardiac sensory neurites associated with afferentneurons within the cardiac neuroaxis.

[0392] Given that bilateral transection of the ansae subclavia abolishesthe neuro-suppressor effects that SCS imparts upon the intrinsic cardiacnervous system (as discussed hereinabove), it appears that thesympathetic nervous system is involved in the effects of SCS on theintrinsic cardiac nervous system. Activation of spinal cord neurons mayinhibit intrinsic cardiac local circuit neurons in a manner similar tothat which occurs when they receive increasing inputs from sympatheticefferent preganglionic neurons (Murphy et al., 1995). Based on theresults obtained during application of SCS to the lumbosacral spinalcord (Linderoth and Foreman, 1999), sympathetic afferent as well asefferent axons may contribute to the suppressor effects that SCS exertson the intrinsic cardiac nervous system.

[0393] Activation of sympathetic efferent preganglionic axons attenuatesthe activity generated by sub-populations of neurons withinintrathoracic ganglia, including those on the heart (Armour, 1991;Murphy et al., 1995). Supramaximal stimulation of sympathetic efferentpreganglionic neurons also leads to a rapid reduction in the capacity ofintrathoracic sympathetic efferent neurons to influence cardiodynamics(Butler et al., 1988). It has been proposed that such suppressor effectsare most likely due to inhibitory synapses within intrathoracic ganglia,including those on the heart (Armour, 1991). In accord with that, spinalcord neurons, when activated, suppress the activity generated byintrinsic cardiac neurons.

[0394] It is known that the activity generated by many intrinsic cardiacneurons increases secondary to transient ventricular ischaemia (Armouret al., 1998). Right atrial neurons are supplied by arterial blood inthe sinoatrial artery arising from the right coronary artery and distalbranches of the circumflex coronary artery (Huang et al., 1993). Sinceocclusion of the left anterior descending coronary artery does notcompromise the arterial blood supply of investigated right atrialneurons (Huang et al., 1993), the effects that regional ventricularischaemia exerted on investigated neurons were primarily the result ofischaemia-induced enhancement of ventricular sensory neurite inputs toidentified neurons rather than any direct effects of ischaemia onidentified somata and/or dendrites (Armour, 1991). Intrinsic cardiacneuronal activity remained elevated throughout the 15-min periods ofregional ventricular ischaemia when performed in the absence of SCS.That these regional coronary artery occlusions affected the ST segmentsof the ECG presumably is reflective of the underlying myocardialischaemia so induced. The processing of cardiac sensory informationwithin the intrinsic cardiac nervous system was affected by SCS, asindicated by changed neuronal responsiveness to chemical (veratridine)activation of their ventricular sensory inputs.

[0395] Given the fact that the capacity of veratridine to affect sensoryneurites associated with cardiac afferent neuron exhibits notachyphylaxis (Thompson et al., 2000), the changed transductionproperties of ventricular sensory inputs to the intrinsic cardiacnervous system is due, in part, to remodeling of the intrinsic cardiacnervous system subsequent to SCS. It should be noted that in clinicalstudies, the sensory effects that SCS imparts persist long after thestimulation has stopped. Patients with refractory angina pectoriscontinue to experience decreased episodes of pain after terminating SCS(Jessurun et al., 1999). Furthermore, the allodynia associated withneuropathic pain can be reduced for as long as 1 h after terminating SCS(Stiller et al., 1996). Application of SCS immediately prior to onset ofLAD occlusion did not blunt the evolution of ischaemic-induced changesin the ECG. It is unlikely that the results obtained by SCS in aclinical setting can be ascribed to alterations in hemodynamics(Hautvast et al., 1998; Linderoth and Foreman, 1999) or coronary arteryblood flow (as discussed hereinabove). This is because SCS exerts itsprimary effects on the intrinsic cardiac nervous system that, in turn,may influence control over regional cardiac electrical or mechanicalevents. These data indicate that activation of spinal cord neuronsinduces a conformational change in the intrinsic cardiac nervous systemthat persists for a considerable period of time after terminating suchactivation. This remodeling of the intrinsic cardiac nervous system canoverride excitatory inputs to it arising from the ischaemic myocardium.Thus, thoracic spinal cord neurons acts to stabilize the intrinsiccardiac nervous system in the presence of ventricular ischaemia andduring reperfusion. The prolonged salutary effects that SCS imparts tosome patients long after it is discontinued is due, in part, toremodeling of the cardiac nervous system.

[0396] Thus it should be apparent that there has been provided inaccordance with the present invention a detailed description, examplesand data showing the SCS or DCA stimulation directly impacts theintrinsic cardiac nervous system and that such an impact can be used tomodify, treat, modulate, suppress, and/or quench the neuronal activityof the intrinsic cardiac nervous system and in turn protect cardiacmyocytes and preserve the electrical stability of the intrinsic cardiacnervous system and the heart itself, that fully satisfies the objectivesand advantages set forth above. Although the invention has beendescribed in conjunction with specific embodiments thereof, it isevident that many alternatives, modifications, and variations will beapparent to those skilled in the art. Accordingly, it is intended toembrace all such alternatives, modifications, and variations that fallwithin the spirit and broad scope of the appended claims.

REFERENCES

[0397] The following references, to the extent that they provideexemplary procedural or other details supplementary to those set forthherein, are specifically incorporated herein by reference in theirentirety as though set forth herein in particular.

[0398] Adamson, P. B., S. S. Hull, Jr., E. Vanoli, G. M. De Ferrari, P.Wisler, R. D. Foreman, A. M. Watanabe, and P. J. Schwartz. Pertussistoxin-induced ADP ribosylation of inhibitor G proteins alters vagalcontrol of heart rate in vivo. Am.J.Physiol 265 (2 Pt 2):H734-H740,1993.

[0399] Adamson, P. B., M. H. Huang, E. Vanoli, R. D. Foreman, P. J.Schwartz, and S. S. Hull. Unexpected interaction between beta-adrenergicblockade and heart rate variability before and after myocardialinfarction; A longitudinal study in dogs at high and low risk for suddencardiac death. Circulation 90: 976-382, 1994.

[0400] Adamson, P. B., E. Vanoli, S. S. Hull, R. D. Foreman, and P. J.Schwartz. Antifibrillatory efficacy of ersentilide, a novelbeta-adrenergic and Ikr blocker, in conscious dogs with a healedmyocardial infarction. Cardiovasc Res. 40 (1):56-63, 1998.

[0401] Allen, T. G. J. and G. Burnstock. Intracellular studies of theelectrophysiological properties of cultured intracardiac neurons of theguinea-pig. J.Physiol. 388: 349-366, 1987.

[0402] Allen, T. G. J. and G. Burnstock. M1 and M2 Muscarinic receptorsmediate excitation and inhibition of guinea-pig intracardiac neurons inculture. J.Physiol. 422: 463-480, 1990.

[0403] Allen, T. G. J., C. J. S. Hassall, and G. Burnstock. MammalianIntrinsic Cardiac Neurons in Cell Culture. In: Neurocardiology, editedby J. A. Armour and J. L. Ardell. New York: Oxford University Press,1994, p. 139-164.

[0404] Ammons, W. S., R. W. Blair, and R. D. Foreman. Vagal afferentinhibition of spinothalamic cell responses to sympathetic afferents andbradykinin in the monkey. Circ.Res. 53 (5):603-612, 1983.

[0405] Ammons, W. S., M. N. Girardot, and R. D. Foreman. Effects ofintracardiac bradykinin on T2-T5 medial spinothalamic cells.Am.J.Physiol 249 (2 Pt 2):R147-R152, 1985.

[0406] Anderson, C., Hole, P., Oxhoj, H. Does pain relief with spinalcord stimulation for angina conceal myocardial infarction? Br Heart J1994; 71:419-421.

[0407] Andresen, M. C. Short-and long-term determinants of baroceptorfunction in aged normotensive and spontaneously hypertensive rats. Circ.Res. 54: 750-759, 1984.

[0408] Applegate, R. J., 1991. Load dependence of left ventriculardiastolic pressure-volume relations during short-term coronary arteryocclusion. Circulation 83, 661-673.

[0409] Ardell, J. L., S. M. Barman, and G. L. Gebber. Sympathetic nervedischarge in chronic spinal cat. Am. J. Physiol. 243: H1463-H1470, 1982.

[0410] Ardell, J. L. and W. C. Randall. Selective vagal innervation ofsinoatrial and atrioventricular nodes in canine heart. Am.J.Physiol.251: H764-H773, 1986.

[0411] Ardell, J. L., W. C. Randall, W. J. Cannon, D. C. Schmacht, andE. Tasdemiroglu. Differential sympathetic regulation ofautomatic,conductile and contractile tissue in dog heart. Am.J.Physiol.255: H1050-H1059, 1988.

[0412] Ardell, J. L., C. K. Butler, F. M. Smith, D. A. Hopkins, and J.A. Armour. Activity of in vivo atrial and ventricular neurons in chronicdecentralized canine hearts. Am.J.Physiol. 260: H713-H721, 1991.

[0413] Ardell, J. L. Structure and Function of Mammalian IntrinsicCardiac Neurons. In Armour, J. A. and J. L. Ardell, eds.Neurocardiology. New York, Oxford University Press. 1994, 95-114.

[0414] Ardell, J. L., 2000. Neurohumoral control of cardiac function.In: Sperelakis,N. (Ed.), Physiology and Pathophysiology of the Heart,4th edn. Kluwer Academic Publishing, Boston, pp. 73-108, Chap. 3.

[0415] Ardell, J. L. Neurohumoral control of cardiac function. HeartPhysiology and Pathophysiology. Academic Press. 2001, 45-59.

[0416] Armour, J. A. Physiological behavior of thoracic cardiovascularreceptors. Amer. J. Physiol. 225:177-185, 1973.

[0417] Armour, J. A. Instant-to-instant reflex cardiac regulation.Cardiology 61:309-328, 1976.

[0418] Armour, J. A. and J. B. Pace. Cardiovascular effects of thoracicafferent nerve stimulation in conscious dogs. Can J. Physiol. Pharmacol.60:1193-1199, 1982.

[0419] Armour, J. A. Synaptic transmission in thoracic autonomic gangliaof the dog. Can. J. Physiol. Pharmacol. 61:793-801, 1983.

[0420] Armour, J. A. Synaptic transmission in chronically decentralizedmiddle cervical and stellate ganglia of the dog. Canadian Journal ofPhysiology and Pharmacology 61, 1149-1155. 1983.

[0421] Armour, J. A. Activity of in situ middle cervical ganglionneurons in dogs, using extracellular recording techniques.Can.J.Physiol.Pharmacol. 63: 704-716, 1985.

[0422] Armour, J. A. Activity of in situ stellate ganglion neurons ofdogs recorded extracellularly. Can.J.Physiol.Pharmacol. 64: 101-111,1986.

[0423] Armour, J. A. Neuronal activity recorded extracellularly inchronic decentralized in situ canine middle cervical ganglia.Can.J.Physiol.Pharmacol. 64: 1038-1046, 1986.

[0424] Armour, J. A. Neuronal activity recorded extracellularly inchronically decentralized in situ canine middle cervical ganglia.Canadian Journal of Physiology and Pharmacology 64, 1038-1046. 1986.

[0425] Armour, J. A. Cardiac effects of electrically inducedintrathoracic autonomic reflexes. Can. J. Physiol. Pharmacol. 66:714-720, 1988.

[0426] Armour, J. A. and R. D. Janes. Neuronal activity recordedextracellularly from in situ mediastinal ganglia. Can. J. Physiol.Pharmacol. 66: 119-127, 1988.

[0427] Armour, J. A. Anatomy and function of the intrathoracic neuronsregulating the mammalian heart. In Zucker, I. H. and J. P. Gilmore, eds.Reflex control of the circulation. Boca Raton, Fla., CRC Press. 1991,1-37.

[0428] Armour, J. A. Intrinsic cardiac neurons.J.Cardiov.Electrophysiol. 2: 331-341, 1991.

[0429] Armour, J. A. Peripheral Autonomic Neurnal Interactions inCardiac Regulation. In Armour, J. A. and J. L. Ardell, eds.Neurocardiology. New York, Oxford University Press. 1994, 219-244.

[0430] Armour, J. A. Histamine-sensitive intrinsic cardiac andintrathoracic extracardiac neurons influence cardiodynamics. AmericanJournal Physiology 270, R906-R913. 1996.

[0431] Armour, J. A. Comparative effects of enodthelin and neurotensinon intrinsic cardiac neurons in situ. Peptides 17, 1047-1052. 1999.

[0432] Armour, J. A. Myocardial ischemia and the cardiac nervous system.Cardiovascular Research 41, 41-54. 1999.

[0433] Armour, J. A., Collier, K., Kember, G., and Ardell, J. L.Differential selectivity of cardiac neurons in separate intrathoracicautonomic ganglia. American Journal Physiology 274, R939-R949. 1998.

[0434] Armour, J. A. and D. A. Hopkins. Activity of canine in situ leftatrial ganglion neurons. Am.J.Physiol. 259: H1207-H1215, 1990.

[0435] Armour, J. A. and D. A. Hopkins. Activity of in vivo ventricularneurons. Am.J.Physiol. 258: H326-H336, 1990.

[0436] Armour, J. A., Huang, M. H., Pelleg, A., and Sylven, C.Responsiveness of in situ canine nodose ganglion cardiac afferentneurons to epicardial mechanoreceptor and/or chemoreceptor stimuli.Cardiovascular Research 28, 1218-1225. 1994.

[0437] Armour, J. A., Huang, M. H., and Smith, F. M. Peptidergicmodulation of in situ canine intrinsic cardiac neurons. Peptides 14,191-202. 1993.

[0438] Armour, J. A., Murphy, D. A., Yuan, B. X., MacDonald, S., andHopkins, D. A. Anatomy of the human intrinsic cardiac nervous system.The Anatomical Record 297, 289-298. 1997.

[0439] Armour, J. A., B. X. Yuan, and C. K. Butler. Cardiac responseselicited by peptide administration to canine intrinsic cardiac neurons.Peptides 11: 753-761, 1990.

[0440] Arora, R. C., J. L. Ardell, and J. A. Armour. Cardiac denervationand cardiac function. Curr.Interv.Cardiol.Rep. 2: 188-195, 2000.

[0441] Augustinsson, L. E., Linderoth, B., Mannheimer, C., Eliasson, T.Spinal cord stimulation in cardiovascular disease. In: Gildenberg P,editor Neurosurgery clinics of North America, 1995, pp. 157-165.

[0442] Augustinsson, L. E., Linderoth, B., Eliasson, T., Mannheimer, C.Spinal cord stimulation in peripheral vascular disease and anginapectoris. In: Gildenberg P, Tasker R, editors, Textbook of stereotacticand functional neurosurgery, NY: McGraw-Hill, 1997, pp. 1973-1978.

[0443] Baan, J., van der Velde, E. T., de Bruin, H. G., Smeenk, G. J.,Koops, J., van Dijk, A. D., Temmerman, D., Senden, J., Buis, B., 1984.Continuousmeasurement of left ventricular volume in animals and humansby conductance catheter. Circulation 70, 812-823.

[0444] Baer, R. W., Payne, B. D., Verrier, E. D., Vlahakes, G. J.,Molodowitch, D., Uhlig, P. N., Hoffman, J. I. E., 1984. Increased numberof myocardial blood flow measurements with radionuclide-labeledmicrospheres. Am. J. Physiol. 246, H418-H434.

[0445] Baker, D. G., H. M. Coleridge, J. C. Coleridge, and T. Nerdrum.Search for a cardiac nociceptor: stimulation by bradykinin ofsympathetic afferent nerve endings in the heart of the cat. J.Physiol306:519-536, 1980.

[0446] Baluk, P. and G. Gabella. Some intrinsic neurons of theguinea-pig heart contain substance P. Neurosci.Lett. 104: 269-273, 1989.

[0447] Baluk, P. and G. Gabella. Some parasympathetic neurons in theguinea-pig heart express aspects of the catecholaminergic phenotype invivo. Cell Tissue Res. 261: 275-285, 1990.

[0448] Barron, K. W., J. E. Croom, C. A. Ray, M. J. Chandler, and R. D.Foreman. Spinal integration of antidromic mediated cutaneousvasodilation during dorsal spinal cord stimulation in the rat.Neurosci.Lett. 260 (3):173-176, 1999.

[0449] Bartfai, T., Iverfeldt, K., and Fisone, G. Regulation and therelease of coexisting neurotransmitters. Ann.Rev.Pharmacol.Toxicol. 28,285-310. 1988.

[0450] Beau, Scott L., Hand, Dwight E., Schuessler, Richard B.,Bromberg, Burt I., Kwon, Brian, Boineau, John P., and Saffitz, JeffreyE. Relative Densities of Muscarinic Cholinergic and B-adrenergicreceptors in the canine sinoatrial node and their relation to sites ofpacemaker activity. Circulation Research 77, 957-963. 1995.

[0451] Besson, J. M. and A. Chaouch. Peripheral and spinal mechanisms ofnociception. Physiol Rev. 67 (1):67-186, 1987.

[0452] Billman G E, Schwartz P I, Stone H L. The effects of dailyexercise on susceptibility to sudden cardiac death. Circulation 1984;69:1182-1189.

[0453] Blair, R. W., R. N. Weber, and R. D. Foreman. Responses ofthoracic spinothalamic neurons to intracardiac injection of bradykininin the monkey. Circ.Res. 51 (1):83-94, 1982.

[0454] Blair, R. W., W. S. Ammons, and R. D. Foreman. Responses ofthoracic spinothalamic and spinoreticular cells to coronary arteryocclusion. J.Neurophysiol. 51 (4):636-648, 1984.

[0455] Blair, R. W., and R. D. Foreman. Activation of feline spinalneurones by potentiated ventricular contractions and other mechanicalcardiac stimuli. J.Physiol 404:649-667, 1988.

[0456] Blomquist, T. M., D. V. Priola, and A. M. Romero. Source ofintrinsic innervation of canine ventricles: a functional study. Am. J.Physiol., 252: H638-H644, 1987.

[0457] Bluemel, K. M., R. D. Wurster, W. C. Randall, M. J. Duff, and M.F. O'Toole. Parasympathetic postganglionic pathways to the sinoatrialnode. Am.J.Physiol. 259: H1504-H1510, 1990.

[0458] Bosnjak, Z. and Kampine, J. P. Cardiac sympathetic afferent cellbodies are located in the peripheral nervous system of the cat.Circulation Research 64, 554-562. 1989.

[0459] Brink, M., de Gasparo, M., Rogg, H., Whitebread, S., and Bullock,G. Localization of Angiotensin II Receptor Subtypes in the Rabbit Heart.Journal Molecular and Cellular Cardiology 27, 459-470. 1995.

[0460] Brodde, Otto-Erich. Beta-adrenoceptors in cardiac disease.Pharmac.Ther. 60, 405-430. 1993.

[0461] Brodde, O. -E. and H. -R. Zerkowski. Neural control of cardiacmyocyte function. In Armour, J. A. and J. L. Ardell, eds.Neurocardiology. New York, Oxford University Press. 1994, 193-218.

[0462] Brown, A. M. Excitation of afferent cardiac sympathetic nervefibres during myocardial ischaemia. J.Physiol 190 (1):35-53, 1967.

[0463] Brown, A. M. Cardiac reflexes. In Berne, R. M., N. Sperelakis,and S. R. Geiger, eds. Handbook of Physiology, The CardiovascularSystem, Section 2, Vol.1, The Heart. Bethesda, American PhysiologicalSociety (Williams and Wilkins). 1979, 677-689.

[0464] Brown, P. B., H. R. Koerber, and R. P. Yezierski.Cross-correlation analysis of connectivities among cat lumbosacraldorsal horn cells. J.Neurophysiol. 42 (5):1199-1211, 1979.

[0465] Burnstock G, Wood J N. Purinergic receptors: their role innociception and primary afferent neurotransmission. Cur Opinion Biol1996;6:526-532.

[0466] Burton, H. and A. D. Loewy. Descending projections from themarginal cell layer and other regions of the monkey spinal cord. BrainRes. 116 (3):485-491, 1976.

[0467] Butler, C. K., Watson-Wright, W. M., Wilkinson, M., Johnston, D.E., Armour, J. A., 1988. Cardiac effects produced by long-termstimulation of acutely decentralized thoracic autonomic ganglia andcardiac nerves: implications for inter-neuronal interactions within thethoracic autonomic nervous system. Can. J. Physiol. Pharmacol. 66,175-184.

[0468] Butler, C. K., F. M. Smith, R. Cardinal, D. A. Murphy, D. A.Hopkins, and J. A. Armour. Cardiac responses to electrical stimulationof discrete loci in canine atrial and ventricular ganglionated plexi.Am.J.Physiol. 259: H1365-H1373, 1990.

[0469] Butler, C. K., F. M. Smith, J. Nicholson, and J. A. Armour.Cardiac effects induced by chemically activated neruons in canineintrathoracic ganglia. Am.J.Physiol. 259: H1108-H1117, 1990.

[0470] Butler, C. K., Watson-Wright, W. M, Wilkinson, M., Johnston, D.E., and Armour, J. A. Cardiac effects produced by long-term stimulationof acutely decentralized thoracic autonomic ganglia and cardiac nerves:implications for inter-neuronal interactions within the thoracicautonomic nervous system. Canadian Journal of Physiology andPharmacology 66, 175-184. 1988.

[0471] Canteras, N. S., S. Chiavegatto, L. E. Valle, and L. W. Swanson.Severe reduction of rat defensive behavior to a predator by discretehypothalamic chemical lesions. Brain Res.Bull. 44 (3):297-305, 1997.

[0472] Cao, J. -M., L. S. Chen, B. H. KenKnight, T. Ohara, M. -H. Lee,J. Tsai, W. W. Lai, H. S. Karagueuzian, P. L. Wolf, M. C. Fishbein, andP. -S. Chen. Nerve sprouting and sudden cardiac death. Circ.Res. 86:816-821, 2000.

[0473] Cao, J. -M., M. C. Fishbein, J. B. Han, W. W. Lai, A. C. Lai, T.-J. Wu, L. Czer, P. L. Wolf, T. A. Denton, P. Shintaku, P. -S. Chen, andL. S. Chen. Relationship between regional hyperinnervation andventricular arrhythmia. Circulation 101: 1960-1969, 2000.

[0474] Carstens, E. and D. L. Trevino. Anatomical and physiologicalproperties of ipsilaterally projecting spinothalamic neurons in thesecond cervical segment of the cat's spinal cord. J.Comp Neurol. 182(1):167-184, 1978.

[0475] Casati, R., F. Lombardi, and A. Malliani. Afferent sympatheticunmyelinated fibres with left ventricular endings in cats. J.Physiol292:135-148, 1979.

[0476] Chahine R, L. Olivia, H. Lockwell and R. Nadeau. Oxygen-freeradicals and myocardial nerve fiber endings. Exp. Toxic Path.46:403-408, 1994.

[0477] Chandler, M. J., S. F. Hobbs, D. C. Bolser, and R. D. Foreman.Effects of vagal afferent stimulation on cervical spinothalamic tractneurons in monkeys. Pain 44 (1):81-87, 1991.

[0478] Chandler, M. J., T. J. Brennan, D. W. Garrison, K. S. Kim, P. J.Schwartz, and R. D. Foreman. A mechanism of cardiac pain suppression byspinal cord stimulation: implications for patients with angina pectoris.Eur.Heart J. 14 (1):96-105, 1993.

[0479] Chandler, M. I., J. Zhang, and R. D. Foreman. Vagal, sympatheticand somatic sensory inputs to upper cervical (C1-C3) spinothalamic tractneurons in monkeys. J.Neurophysiol. 76 (4):2555-2567, 1996.

[0480] Chandler, M. J., J. Zhang, C. Qin, Y. Yuan, and R. D. Foreman.Intrapericardiac injections of algogenic chemicals excite primate C1-C2spinothalamic tract neurons. Am.J.Physiol Regul.Integr.Comp Physiol 279(2):R560-R568, 2000.

[0481] Chang, Y., S. R. Stover, and D. B. Hoover. Regional localizationand abundance of calcitonin gene-related peptide receptors in guinea pighearts. J.Mol.Cell.Cardiol. 33: 745-754, 2001.

[0482] Chapleau, M. W., Cunningham, J. T., Sullivan, M. J., Wachtel, R.E., and Abboud, F. M. Structural versus functional modulation of thearterial baroreflex. Hypertension 26, 341-347. 1995.

[0483] Chauhan, A., Mullins, P. A., Thuraisingham, S. I., Taylor, G.,Petch, M. C.,Schofield, P. M., 1994. Effect of transcutaneous electricalnerve stimulation on coronary blood flow. Circulation 89, 921-926.

[0484] Cardinal, R., P. Savard, J. A. Armour, R. Nadeau, D. L. Carson,and A. R. LeBlanc. Mapping of ventricular tachycardia induced bythoracic neural stimulation in dogs. Can.J.Physiol Pharmacol. 64(4):411-418, 1986.

[0485] Cardinal, R., B. J. Scherlag, M. Vermeulen, and J. A. Armour.Distinct activation patterns of idioventricular rhythms andsympathetically-induced ventricular tachycardias in dogs withatrioventricular block. Pacing Clin.Electrophysiol. 15 (9):1300-1316,1992.

[0486] Cardinal, R., R. Nadeau, C. Laurent, G. Boudreau, and J. A.Armour. Reduced capacity of cardiac efferent sympathetic neurons torelease noradrenaline and modify cardiac function in tachycardia-inducedcanine heart failure. Can.J.Physiol Pharmacol. 74 (9):1070-1078, 1996.

[0487] Chen, P. -S., L. S. Chen, B. Sharifi, H. S. Karagueuzian, and M.C. Fishbein. Sympathetic nerve sprouting, electrical remodeling and themechanisms of sudden cardiac death. Cardiov.Res. 50: 409-416, 2001.

[0488] Cheng, Z., Powley, T. L., Schwaber, J. S., and Doyle, F. J. Vagalafferent innervation of the atria of the rat heart reconstructed withconfocal microscopy. The Journal of Comparative Neurology (381), 1-17.1997.

[0489] Cole, C. R., E. H. Blackstone, F. J. Pashkow, C. E. Snader, andM. S. Lauer. Heart-rate recovery immediately after exercise as apredictor of mortality. N.Engl.J.Med. 341 (18):1351-1357, 1999.

[0490] Coyle, J. T., M. E. Molliver, and M. J. Kuhar. In situ injectionof kainic acid: a new method for selectively lesioning neural cellbodies while sparing axons of passage. J.Comp Neurol. 180 (2):301-323,1978.

[0491] Craig, Jr., A. D., and D. N. Tapper. Lateral cervical nucleus inthe cat: functional organization and characteristics. J.Neurophysiol. 41(6):1511-1534, 1978.

[0492] Croom, J. E., Foreman, R. D., Chandler, M. J., and Barron, K. W.Cutaneous vasodilation during dorsal column stimulation is mediated bydorsal roots and CGRP. American Journal Physiology 272, H950-H957. 1997.

[0493] Crowe, R. and G. Burnstock. Fluorescent histochemicallocalization of quinacrine-postive neurons in the guinea-pig and rabbitatrium. Cardiov.Res. 16: 384-390, 1982.

[0494] Curtis, M. J., Hearse, D. J., 1989. Reperfusion-inducedarrhythmias are critically dependent upon occluded zone size: relevanceto the mechanism of arrhythmogenesis. J. Mol. Cell Cardiol. 21, 625-637.

[0495] Dalsgaard, C. J., A. Franco-Cereceda, A. Saria, J. M. Lundberg,E. Theodorsson-Norheim, and T. Hökfelt. Distribution and origin ofsubstance P- and neuropeptide Y-immunoreactive nerves in the guinea-pigheart. Cell Tissue Res. 243: 477-485, 1986.

[0496] Darvesh, S., D. M. Nance, D. A. Hopkins and J. A. Armour.Distribution of neuropeptide immunoreactivity in intact and chronicallydecentralized middle cervical and stellate ganglia of dogs. J. AutonomicNerv. Syst., 21: 167-180, 1987.

[0497] Dávila-Garcia, M. I., J. L. Musachio, D. C. Perry, Y. Xiao, A.Horti, E. D. London, R. F. Dannals, and K. J. Kellar. [¹²⁵]IPH, anepibatidine analog, binds with high affinity to neuronal nicotiniccholinergic receptors. J.Pharmacol.Exp.Ther. 445-451, 1997.

[0498] Della, N. G., R. E. Papka, J. B. Furness, and M. Costa.Vasoactive intestinal peptide-like immunoreactivity in nerves associatedwith the cardiovascular system of guinea-pigs. Neurosci. 9: 605-619,1983.

[0499] De Ferrari, G. M., M. Mantica, E. Vanoli, S. S. Hull, Jr., and P.J. Schwartz. Scopolamine increases vagal tone and vagal reflexes inpatients after myocardial infarction. J.Am.Coll.Cardiol. 22 (5):1327-1334, 1993.

[0500] DeJongste, M. J. L., Haaksma, J., Hautvast, R. W., Hillege, H.L., Meyler, P. W, Staal, M. J., Sanderson, J. E., Lie, K. I., 1994a.Effects of spinal ord stimulation on daily life myocardial ischemia inpatients with severe coronary artery disease. A prospective ambulatoryECG study. Br. Heart J. 71, 413-418.

[0501] DeJongste, M. J. L., Hautvast, R. W. M., Hillege, H., Lie, K. I.,1994b. Efficacy of spinal cord stimulation as an adjuvant therapy forintractable angina pectoris: a prospective randomized clinical study. J.Am. Coll. Cardiol. 23, 1592-1597.

[0502] DeJongste, M. J., Nagelkerke, D., Hooyschuur, C. M. Journe, H. L.Meyler P. W. Staal M J, De Jonge P., Lie, K. I. Stimulationcharacteristics, complications, and efficacy of spinal cord stimulationsystems in patients with refractory angina: a prospective feasabilitystudy. Pacing Clin Electrophysiol 1994; 17:1751-1760.

[0503] De Landsheere, C., Mannheimer, C., Habets, A., Guillaume, M.,Bourgeois, I., Augustinsson, L. -E., Eliasson, T., Lamotte, D.,Kulbertus, H., Rigo, P., 1992. Effect of spinal cord stimulation onregional myocardial perfusion assessed by positron emission tomography.Am. J. Cardiol. 69, 1143-1149.

[0504] Dole, W. P., Bishop, V. S., 1982. Influence of autoregulation andcapacitance on diastolic coronary artery pressure-flow relationships inthe dog. Circ. Res. 51, 261-270.

[0505] Eblen-Zajjur, A. A., and J. Sandkuhler. Synchronicity ofnociceptive and non-nociceptive adjacent neurons in the spinal dorsalhorn of the rat: stimulus-induced plasticity. Neuroscience 76 (1):39-54,1997.

[0506] Eliasson, T., Augustinsson, L. E., and Mannheimer, C. Spinal cordstimulation in severe angina pectoris: Presentation of current studies,indications and clinical experience. Pain 65, 169-179. 1996.

[0507] Eliasson, T., Mannheimer, C., Waagstein, F., Andersson, B., Berg,C. H., Augustinsson, L. E., Hedner, T., and Larsson, G. Myocardialturnover of endogenous opoids and CGRP in the human heart and theeffects of spinal cord stimulation. Cardiology 89, 170-177. 1998.

[0508] Ellison, J. P. and R. G. Hibbs. An ultrastructural study ofmammalian cardiac ganglia. J.Mol.Cell.Cardiol. 8: 89-101, 1976.

[0509] Euchner-Wamser, S. T. Meller, and G. F. Gebhart. A model ofcardiac nociception in chronically instrumented rats: behavioral andelectrophysiological effects of pericardial administration of algogenicsubstances. Pain 58 (1):117-128, 1994.

[0510] Farrell, D. M., C. C. Wei, J. Tallaj, J. L. Ardell, J. A. Armour,G. R. Hageman, W. E. Bradley, and L. J. Dell'Italia. Angiotensin IImodulates catecholamine release into interstitial fluid of canineventricle in vivo. Am.J.Physiol.Heart Circ.Physiol. 281: H813-H822,2001.

[0511] Farrell, D. M., C. C. Wei, J. L. Ardell, J. A. Armour, G. R.Hageman, W. E. Bradley and L. J. Dell'Italia. Angiotensin II modulatesnorepinephrine release into the interstitial fluid of the caninemyocardium in vivo. Am. J. Physiol., in press, 2001.

[0512] Farrell, T. G., O. Odemuyiwa, Y. Bashir, T. R. Cripps, M. Malik,D. E. Ward, and A. J. Camm. Prognostic value of baroreflex sensitivitytesting after acute myocardial infarction. Br.Heart J. 67 (2):129-137,1992.

[0513] Fee, J. D., W. C. Randall, R. D. Wurster, and J. L. Ardell.Selective ganglionic blockade of vagal inputs to sinoatrial and/oratrioventricular regions. J.Pharmacol.Exp.Ther. 242: 1006-1012, 1987.

[0514] Ferrer, E. Lopez, R. Blanco, R. Rivera, J. Krupinski, and E.Marti. Differential c-Fos and caspase expression following kainic acidexcitotoxicity. Acta Neuropathol.(Berl) 99 (3):245-256, 2000.

[0515] Flink, R., and J. Westman. Different neuron populations in thefeline lateral cervical nucleus: a light and electron microscopic studywith the retrograde axonal transport technique. J.Comp Neurol. 250(3):265-281, 1986.

[0516] Flink, R. and B. A. Svensson. Fluorescent double-labelling studyof ascending and descending neurones in the feline lateral cervicalnucleus. Exp.Brain Res. 62 (3):479-485, 1986.

[0517] Foreman, R. D. Spinal cord neuronal regulation of thecardiovascular system. In Armour, J. A. and J. L. Ardell, eds.Neurocardiology. New York, Oxford University Press. 1994, 245-276.

[0518] Foreman, R. D. Mechanisms of cardiac pain. Annu.Rev.Physiol. 61,143-167. 1999.

[0519] Foreman, R. D., R. W. Blair, H. R. Holmes, and J. A. Armour.Correlation of ventricular mechanosensory neurite activity withmyocardial sensory field deformation. Am.J.Physiol 276 (4 Pt2):R979-R989, 1999.

[0520] Foreman, R. D., R. W. Blair, H. R. Holmes and J. A. Armour.Correlation of activity generated by sympathetic afferent ventricularmechanosensory neurites with sensory field deformation in the normal andischemic myocardium. Am. J. Physiol., 276: R976-R989, 1999.

[0521] Foreman, R. D., B. Linderoth, J. L. Ardell, K. W. Barron, M. J.Chandler, S. S. Hull, G. J. TerHorst, M. J. L. DeJongste, and J. A.Armour. Modulation of intrinsic cardiac neurons by spinal cordstimulation: implications for therapeutic use in angina pectoris.Cardiov.Res. 47: 367-375, 2000.

[0522] Fu, Q. G., M. J. Chandler, D. L. McNeill, and R. D. Foreman.Vagal afferent fibers excite upper cervical neurons and inhibit activityof lumbar spinal cord neurons in the rat. Pain 51 (1):91-100, 1992.

[0523] Furukawa, Y., D. W. Wallick, M. D. Carlson, and P. J. Martin.Cardiac electrical responses to vagal stimulation of fibers to discretecardiac regions. Am.J.Physiol. 258: H1112-H1118, 1990.

[0524] Furukawa, Y., D. W. Wallick, P. J. Martin, and M. N. Levy.Chronotropic and dromotropic responses to stimulation of intracardiacsympathetic nerves to sinoatrial or atrioventricular nodal region inanesthetized dogs. Circ.Res. 66: 1391-1399, 1990.

[0525] Gagliardi, M., W. C. Randall, D. Bieger, R. D. Wurster, D. A.Hopkins, and J. A. Armour. Activity of in vivo canine cardiac plexusneurons. Am.J.Physiol. 255: H789-H800, 1988.

[0526] Gebber, G. L., S. Zhong and Y. Paitel. Bispectral analysis ofcomplex patterns of sympathetic nerve discharge. Am.J. Physiol. 271:R1173-R1185, 1996.

[0527] Glickstein, S. B., E. V. Golanov, and D. J. Reis. Intrinsicneurons of fastigial nucleus mediate neurogenic neuroprotection againstexcitotoxic and ischemic neuronal injury in rat. J.Neurosci. 19(10):4142-4154, 1999.

[0528] Grill, H. J., M. I. Friedman, R. Norgren, G. Scalera, and R.Seeley. Parabrachial nucleus lesions impair feeding response elicited by2,5-anhydro-D-mannitol. Am.J.Physiol 268 (3 Pt 2):R676-R682, 1995.

[0529] Gu, J., J. M. Polak, J. M. Allen, W. M. Huang, M. N. Sheppard, K.Tatemoto, and S. R. Bloom. High concentrations of a novel peptideneuropeptide Y, in the innervation of mouse and rat heart.J.Histochem.Cytochem. 32: 467-472, 1984.

[0530] Hancock, J. C., D. B. Hoover, and M. W. Hougland. Distribution ofmuscarinic receptors and acetylcholinesterase in the rat heart.J.Auton.Nerv.Syst. 19: 59-66, 1987.

[0531] Harding, S. E., L. A. Brown, D. G. Wynne, C. H. Davies, and P. A.Poole-Wilson. Mechanisms of β adrenoceptor desensitization in thefailing human heart. Cardiov. Res. 28: 1451-1460, 1994.

[0532] Hassall, C. J. S. and G. Burnstock. Intrinsic neurons andassociated cells of the guinea-pig heart in culture. Brain Res. 364:102-113, 1986.

[0533] Hassall, C. J. S. and G. Burnstock. Immunocytochemicallocalization of neuropeptide Y and 5-hydroxytryptamine in asubpopulation of amine-handling intracardiac neurons that do not containdopamine β-hydroxylase in tissue culture. Brain Res. 422: 74-82, 1987.

[0534] Hautvast, R. W., Blanksma, P. K., DeJongste, M. J., Pruim, J.,Van der Wall, E. E., Vaalberg, W., Lie, K. I., 1996. Effect of spinalcord stimulation on myocardial blood flow assessed by positron emissiontomography in patients with refractory angina pectoris. Am. J. Cardiol.77, 462-467.

[0535] Hautvast, R. W. M., Ter Horst, G. J., DeJong, B. M., DeJongste,M. J., Blanksma, P. K., Paans, A. M., Korf, J., 1997. Relative changesin regional cerebral blood flow during spinal cord stimulation inpatients with refractory angina pectoris. Eur. J. Neurosci. 9,1178-1183. Hautvast, R. W., DeJongste, M. J. L., Staal, M. J., VanGilst, V. H., and Lie, K. I. Spinal cord stimulation in chronicintractable angina pectoris: a randomized, controlled efficacy study.American Heart Journal 136, 114-120. 1998.

[0536] Herrera, D. G., and H. A. Robertson. Activation of c-fos in thebrain. Prog Neurobiol. 50 (2-3):83-107, 1996.

[0537] Hillarp, N. -A., Peripheral autonomic mechanisms. In: Handbook ofPhysiology, Section I: Neurophysiology. ed. J.Field, AmericanPhysiological Society, Washington, 1960, pp 979-1006.

[0538] Hobbs, S. F., U. T. Oh, M. J. Chandler, and R. D. Foreman.Cardiac and abdominal vagal afferent inhibition of primate T9-S1spinothalamic cells. Am.J.Physiol 257 (4 Pt 2):R889-R895, 1989.

[0539] Hobbs, S. F., M. J. Chandler, D. C. Bolser, and R. D. Foreman.Segmental organization of visceral and somatic input onto C3-T6spinothalamic tract cells of the monkey. J.Neurophysiol. 68(5):1575-1588, 1992.

[0540] Hoffman, J. I. E., 1987. A critical view of coronary reserve.Circulation 75 {haeck over (Z)}. Suppl. I, 1-6.

[0541] Holland, R. P. and H. Brooks. TQ-ST segment mapping: Criticalreview and analysis of current concepts. Am.J.Cardiol. 40: 110-129,1977.

[0542] Hoover, D. B. and J. C. Hancock. Distribution of substance Pbinding sites in guinea-pig heart and pharmacological effects ofsubstance P. J.Auton.Nerv.Syst. 23: 189-197, 1988.

[0543] Hoover, D. B., R. H. Baisden, and S. X. Xi-Moy. Localization ofMuscarinic Receptor mRNAs in Rat Heart and Intrinsic Cardiac Ganglia byIn Situ Hybridization. Circ. Res. 75: 813-820, 1994.

[0544] Hoover, D. B., Y. Chang, J. C. Hancock, and L. Zhang. Actions oftachykinins within the heart and their relevance to cardiovasculardisease. Jpn.J.Pharmacol. 84: 367-373, 2000.

[0545] Hopkins, D. A. and Armour, J. A. Ganglionic distribution ofafferent neurons innervating the canine heart and physiologicalidentified cardiopulmonary nerves. Journal of the Autonomic NervousSystem 26, 213-222. 1989.

[0546] Hopkins, D. A. and H. H. Ellenberger. Cardiorespiratory neuronsin the medulla oblongata: input and output relationships. In Armour, J.A. and J. L. Ardell, eds. Neurocardiology. New York, Oxford UniversityPress. 1994, 277-308.

[0547] Hopkins, D. A., S. MacDonald, D. A. Murphy, and J. A. Armour.Pathology of intrinsic cardiac neurons from ischemic human hearts. AnatRec 259: 424-436, 2000.

[0548] Horackova, M. and J. A. Armour. Role of peripheral autonomicneurones in maintaining adequate cardiac function. Cardiov. Res. 30:326-335, 1995.

[0549] Horackova, M. and Armour, J. A. ANG II modifies cardiomyocytefunction via extracardiac and intracardiac neurons: in situ and in vitrostudies. American Journal Physiology 272, R766-R775. 1997.

[0550] Horackova, M., J. A. Armour and Z. Byczko. Multiple neurochemicalcoding of intrinsic cardiac neurons in whole-mount guinea-pig atria;confocal microscopic study. Cell Tissue Res., 297: 409-421, 1999.

[0551] Huang, B., T. El-Sherif, M. Gidh-Jain, D. Qin, and N. El-Sherif.Alterations of sodium channel kinetics and gene expression in thepostinfarction remodeled myocardium. J.Cardiov.Electrophysiol. 12:226-238, 2001.

[0552] Huang H S, Pan H, Stahl G L, Longhurst I C. Ischemia- andreperfusion-sensitive cardiac sympathetic afferents: influence of H₂O₂and hydroxyl radicals. Am. J. Physiol. 1995:269:H888-H901.

[0553] Huang H S, Stahl G L, Longhurst I C. Cardiac-cardiovascularreflexes induced by hydrogen peroxide in cats. Am J Physiol1995;268:H2114-2124.

[0554] Huang, M. H., J. L. Ardell, B. D. Hanna, S. G. Wolf, and J. A.Armour. Effects of transient coronary artery occlusion on canineintrinsic cardiac neuronal activity. Integ.Physiol.Behav.Sci. 28: 5-21,1993.

[0555] Huang, M. H., F. M. Smith, and J. A. Armour. Amino acids modifyactivity of canine intrinsic cardiac neurons involved in cardiacregulation. Am.J.Physiol. 264: H1275-H1282, 1993.

[0556] Huang, M. H., F. M. Smith, and J. A. Armour. Modulation of insitu canine intrinsic cardiac neuronal activity by nicotinic, muscarinicand β-adrenergic agonists. Am.J.Physiol. 265: R659-R669, 1993.

[0557] Huang, M. H., C. Sylven, A. Pelleg, F. M. Smith, and J. A.Armour. Modulation of in situ canine intrinsic cardiac neuronal activityby local applied adenosine,ATP or their analogs. Am.J.Physiol. 265:R914-R922, 1993.

[0558] Huang, M. H., Wolf, S. G., and Armour, J. A. Ventriculararrhythmias induced by chemically modified intrinsic cardiac neurones.Cardiovascular Research 28, 636-642. 1994.

[0559] Huang, M. H., C. Sylvén, M. Horackova and J. A. Armour.Ventricular sensory neurons in canine dorsal root ganglia: effects ofadenosine and substance P. Am. J. Physiol., 269: R318-R324, 1995.

[0560] Huang, M. H., Negoescu, R. M., Horackova, M., Wolf, S. G., andArmour, J. A. Polysensory response characteristics of dorsal rootganglion neurons that may serve sensory functions during myocardialischemia. Cardiovascular Research 32, 503-515. 1996.

[0561] Hull S S Jr, Evans E, Vanoli E, Adamson P B, Yeo C, Albert D E,Stramba-Babiale M, Foreman R D, Schwartz P J: Heart Rate VariabilityBefore and After Myocardial Infarction in Dogs at High and Low risk forsudden cardiac death. J Am Coll Cardiol 1990; 16; 978-985.

[0562] Hull S S Jr, Vanoli E, Adamson P B, Verrier R L, Foreman R D,Schwartz P J. Exercise training confers anticipatory protection fromsudden death during acute myocardial ischemia. Circulation1994;89:548-552.

[0563] Hull, S. S., E. Vanoli, P. B. Adamson, G. M. De Ferrari, R. D.Foreman, and P. J. Schwartz. Do increases in markers of vagal activityimply protection from sudden cardiac death? The case of scopolamine.Circulation 91: 2516-2519, 1995.

[0564] Jacobowitz, D. Histochemical studies of the relationship ofchromaffin cells and adrenergic nerves fibers to the cardiac ganglia ofseveral species. J.Pharmacol.Exp.Ther. 158: 227-240, 1967.

[0565] Jacobowitz, D., T. Cooper, and H. B. Barner. Histochemical andchemical studies of the localization of adrenergic and cholinergicnerves in normal and denervated cat hearts. Circ.Res. 20: 289-298, 1967.

[0566] Jakobs, K. H., Minuth, M., Bauer, S., Grandt, R., Greiner, C.,and Zubin, P. Dual regulation of adenylate cyclase. A signaltransduction mechanism of membrane receptors. Basic Res.Cardiol. 81,1-9. 1986.

[0567] Jessurun, G. A. J., Meeder, J. G., and DeJongste, M. J. L. PainReview 4, 89-99. 1997.

[0568] Jessurun, G. A., Tio, R. A., DeJongste, M. J. L., Hautvast, R.W., Den Heijer, P., Crijns, H. J., 1998. Coronary blood flow dynamicsduring transcutaneous electrical stimulation for stable angina pectorisassociated with severe narrowing of one major coronary artery. Am. J.Cardiol. 82, 921-926.

[0569] Jessurun, G. A., DeJongste, M. J. L., Hautvast, R. W., Tio, R.A., Brouwer, J., vanLelieveld, S., Crijns, H. J., 1999. Clinicalfollow-up after cessation of chronic electrical neuromodulation inpatients with sever coronary artery disease: a prospective randomizedcontrolled study on putative involvement of sympathetic activity. PacingClin. Electrophysiol. 22, 1432- 1439.

[0570] Katchanov G, Xu J, Clay A, Pelleg A.Electrophysiological-anatomical correlates of ATP-triggered vagal reflexin the dog. IV. Role of LV vagal afferents. Am J Physiol1997;272:H1898-H1903.

[0571] Katz, D. M., and H. J. Karten. Substance P in the vagal sensoryganglia. Localization in cell bodies and pericellular arborizations. J.Comp. Neurol. 193:549-564, 1980.

[0572] Kember, G. C., G. A. Fenton, K. Collier and J. A. Armour.Stochastic resonance in a hysteretic population of cardiac neurons.Physical Rev. E, 61: 1816-1824, 2000.

[0573] Kember, G. C., G. A. Fenton, J. A. Armour and N. Kalyaniwalla. Acompetition model for aperiodic stochastic resonance in a Fitz-HughNagumo model of cardiac sensory neurons. Physical Rev. E, 63: 041911,1-6, 2001.

[0574] Kingma Jr., J. G., Armour, J. A., Rouleau, J. R., 1994. Chemicalmodulation of in situ cardiac neurones influences myocardial blood flowin the anesthetized dog. Cardiovasc. Res. 28, 1403-1406.

[0575] Kingma, J. G., B. Linderoth, J. L. Ardell, J. A. Armour, M. J. L.DeJongste, and R. D. Foreman. Neuromodulation therapy does not influenceblood flow distribution or left-ventricular dynamics during acutemyocardial ischemia. Autonomic Neuroscience: Basic and Clinical 91:47-54, 2001.

[0576] Klèber, A. G., M. J. Janse, M. J. L. van Capelle, and D. Durrer.Mechanisms and time course of T-Q and S-T segment changes during acuteregional myocardial ischemia in the pig heart determined byextracellular and intracellular recording. Circ.Res. 42: 603-613, 1978.

[0577] Kleiger, R. E., J. P. Miller, J. T. Bigger, Jr., and A. J. Moss.Decreased heart rate variability and its association with increasedmortality after acute myocardial infarction. Am.J.Cardiol. 59(4):256-262, 1987.

[0578] Kocsis, B. Basis for differential coupling between rhythmicdischarges of sympathetic efferent nerves. Amer. J. Physiol. 267,R1008-R1019, 1994.

[0579] Kocsis, B., T, Karlsson and B. G. Wallin. Cardiac-abdnoncardiac-related coherence between sympathetic drives to muscles ofdifferent hind limbs. Am. J. Physiol. 276: R1608-R1616, 1999.

[0580] Kompa, A. R., Molenaar, P., and Summers, R. J. Effect of chemicalsympathectomy on (−)-isoprenaline-induced changes in cardiacbeta-adrenoceptor subtypes in the guinea-pig and rat. Journal AutonomicPharmacology 14, 411-423. 1994.

[0581] Kovacs, K. J.. c-Fos as a transcription factor: a stressful(re)view from a functional map. Neurochem.Int. 33 (4):287-297, 1998.

[0582] Kumada, T., K. Gallagher, A. Battler, F. White, W. S. Kemper, andJ. Jr. Ross. Comparison of post-pacing and exercise-induced myocardialdysfunction during collateral development in conscious dogs. Circulation65: 1178-1185, 1982.

[0583] Kuntz, A. The Autonomic Nervous System. Philadelphia, Lea andFebiger. 1934.

[0584] Kuo, D. C., J. J. Oravitz, and W. C. DeGroat. Tracing of afferentand efferent pathways in the left inferior cardiac nerve of the catusing retrograde and transganglionic transport of horseradishperoxidase. Brain Res. 321 (1):111-118, 1984.

[0585] Kuypers, F. A., R. A. Lewis, M. Hua, M. A. Schott, D. Discher, J.D. Ernst, and B. H. Lubin. Detection of altered membrane phospholipidasymmetry in subpopulations of human red blood cells using fluorescentlylabeled annexin V. Blood 87 (3):1179-1187, 1996.

[0586] La Rovere, M. T., J. T. Bigger, F. Marcus, A. Mortara, and P. J.Schwartz. ATRAMI (Autonomic Tone and Reflexes After MyocardialInfarction). Lancet 351: 478-484, 1998.

[0587] La Rovere M. T., Specchia G, Mortara A, Schwartz P J: Baroreflexsensitivity, clinical correlates and cardiovascular mortality amongpatients with a first myocardial infarction: a prospective study.Circulation 1988; 78:816-24.

[0588] La Rovere, M. T., G. D. Pinna, S. H. Hohnloser, F. I. Marcus, A.Mortara, R. Nohara, J. T. Bigger, Jr., A. J. Camm, and P. J. Schwartz.Baroreflex Sensitivity and Heart Rate Variability in the Identificationof Patients at Risk for Life-Threatening Arrhythmias: Implications forClinical Trials. Circulation 103 (16):2072-2077, 2001.

[0589] Lathrop, D. A. and P. M. Spooner. On the neural connection.J.Cardiov.Electrophysiol. 12: 841-844, 2001.

[0590] Laurent, C. E., R. Cardinal, G. Rousseau, M. Vermeulen, C.Bouchard, M. Wilkinson, J. A. Armour, and M. Bouvier. Functionaldesensitization to isoproterenol without reducing cAMP production incanine failing cardiocytes. Am.J.Physiol Regul.Integr.Comp Physiol 280(2):R355-R364, 2001.

[0591] Levett, J. M., Murphy, D. A., McGuirt, A. S., Ardell, J. L., andArmour, J. A. Cardiac augmentation can be maintained by continuousexposure of intrinsic cardiac neurons to a beta-adrenergic agonist orangiotensinII.J.Surg.Res.66,167-173.1996.

[0592] Levitzki, Alexander. Regulation of adenylate cyclase by hormonesand G-proteins. FEBS Letters 211(2), 113-118. 1987.

[0593] Levy, M. N. and M. R. Warner. Parasympathetic Effects on CardiacFunction. In Armour, J. A. and J. L. Ardell, eds. Neurocardiology.Oxford University Press. 1994, 53-76.

[0594] Lewis, C. D., G. L. Gebber, P. D. Larsen and S. M. Barman.Long-term correlations in the spike trains of medullary sympatheticneurons. J. Neurophysiol. 85: 1614-1623, 2001.

[0595] Li, B. H., A. C. Spector, and N. E. Rowland. Reversal ofdexfenfluramine-induced anorexia and c-Fos/c-Jun expression by lesion inthe lateral parabrachial nucleus. Brain Res. 640 (1-2):255-267, 1994.

[0596] Linden, R. J. and C. T. Kappagoda. Atrial Receptors. CambridgeUniv. Press, Cambridge, England, 1982.

[0597] Linderoth, B., Fedorcsak I, Meyerson, B. A. Peripheralvasodilatation after spinal cord stimulation: animal studies of putativeeffector mechanisms. Neurosurgery 1991; 28:187-195.

[0598] Linderoth, B., L. Gunasekera, and B. A. Meyerson. Effects ofsympathectomy on skin and muscle microcirculation during dorsal columnstimulation: animal studies. Neurosurgery 29 (6):874-879, 1991.

[0599] Linderoth, B., P. Herregodts, and B. A. Meyerson. Sympatheticmediation of peripheral vasodilation induced by spinal cord stimulation:animal studies of the role of cholinergic and adrenergic receptorsubtypes. Neurosurgery 35 (4):711-719, 1994.

[0600] Linderoth, B., and R. D. Foreman. Physiology of spinal cordstimulation. Review and update. Neuromodulation 2: 150-164, 1999.

[0601] Lombardi, F., Bella P. Della, R. Casati, and A. Malliani. Effectsof intracoronary administration of bradykinin on the impulse activity ofafferent sympathetic unmyelinated fibers with left ventricular endingsin the cat. Circ.Res. 48 (1):69-75, 1981.

[0602] Lundberg, J. M., A. Franco-Cereceda, X. Hua, T. Hokfelt, and J.A. Fisher. Co-existance of substance P and calcitonin gene-relatedpeptide-like immunoreactivities in sensory nerves in relation tocardiovascular and bronchoconstrictor effects of capsaicin.Eur.J.Pharmacol. 108: 315-319, 1985.

[0603] Macdonald R L, Skerrit J H, Werz M A. Adenosine Agonists ReduceVoltage-Dependent Calciumconductance of Mouse Sensory Nuerones in CellCulture. J PHYSIOL LOND 1986; 370:75-90.

[0604] Maixner, W., K. B. Touw, M. J. Brody, G. F. Gebhart, and J. P.Long. Factors influencing the altered pain perception in thespontaneously hypertensive rat. Brain Res. 237 (1):137-145, 1982.

[0605] Maixner, W., and A. Randich. Role of the right vagal nerve trunkin antinociception. Brain Res. 298 (2):374-377, 1984.

[0606] Malik M, Camm A J. Components of heart rate variability: whatthey really mean and what they really measure. Am J Cardiol1993;72:821-822.

[0607] Malliani, A., G. Recordati, and P. J. Schwartz. Nervous activityof afferent cardiac sympathetic fibres with atrial and ventricularendings. J.Physiol 229 (2):457-469, 1973.

[0608] Malliani, A. Cardiovascular sympathetic afferent fibers. Rev.Physiol. Biochem. Pharmacol. 94:11-74, 1982.

[0609] Mannheimer, C., Carlsson, C. -A., Emanuelsson, H., Vendin, A.,Waagstein, F., Wilhelmsson, C., 1985. The effects of transcutaneouselectric nerve stimulation in patients with severe angina pectoris.Circulation 71, 308-316.

[0610] Mannheimer, C., Eliasson, T., Andersson, B., Berg, C. H.,Augustinsson, L. E., Emanuelsson, H., and Waagstein, F. Effects ofspinal cord stimulation in angina pectoris induced by pacing andpossible mechanisms of action. British Medical Journal 307, 477-480.1993.

[0611] Mannheimer, C., Eliasson, T., Augustinsson, L. E., Blomstrand,C., Emanuelsson, H., Larsson, S., Norrsell, H., and Hjalmarsson, A.Electrical stimulation versus coronary bypass surgery in severe anginapectoris. The ESBY study. Circulation 97, 1157-1163. 1998.

[0612] Massari, V. J., Johnson, T. A., and Gatti, P. J. Cardiotopicorganization of the nucleus ambiguus? An anatomical and physiologicalanalysis of neurons regulating atrioventicular conduction. BrainResearch 679, 227-240. 1995.

[0613] Matsushita, M. Ascending propriospinal afferents to area X(substantia grisea centralis) of the spinal cord in the rat. Exp.BrainRes. 119 (3):356-366, 1998.

[0614] McGuirt, A. S., Schmacht, D. C., and Ardell, J. L. Autronomicinteractions for control of atrial rate are maintained after SA nodalparasympathectomy. American Journal Physiology 272, H2525-H2533. 1997.

[0615] McLennan, H., and D. Lodge. The antagonism of amino acid-inducedexcitation of spinal neurones in the cat. Brain Res. 169 (1):83-90,1979.

[0616] Meller, S. T., and G. F. Gebhart. A critical review of theafferent pathways and the potential chemical mediators involved incardiac pain. Neuroscience 48 (3):501-524, 1992.

[0617] Melssen, W. J., and W. J. Epping. Detection and estimation ofneural connectivity based on crosscorrelation analysis. Biol.Cybern. 57(6):403-414, 1987.

[0618] Melzack, R., Wall, P. D., 1965. Pain mechanisms: a new theory.Science 150, 971-979.

[0619] Messina, L. M., Hanley, F. L., Uhlig, P. N., Baer, R. W.,Grattan, M. T., Hoffman, J. I. E., 1985. Effects of pressure gradientsbetween branches of the left coronary artery on the pressure axisintercept and the shape of steady state circumflex pressure-flowrelations in dogs. Circ. Res. 56, 11-19.

[0620] Middlekauff, H. R., S. A. Rivkees, H. E. Raybould, B. Bitticaca,J. I. Goldhaber and J. N. Weiss. Localization and functional effects ofadenosine A1 receptors on cardiac vagal afferents in adult rats. Am. J.Physiol. 274:H441-H447, 1998.

[0621] Miller, K. E., V. D. Douglas, A. B. Richards, M. J. Chandler, andR. D. Foreman. Propriospinal neurons in the C1-C2 spinal segmentsproject to the L5-S1 segments of the rat spinal cord. Brain Res.Bull. 47(1):43-47, 1998.

[0622] Miwa, H., T. Fuwa, K. Nishi, and Y. Mizuno. Effects of the globuspallidus lesion on the induction of c-Fos by dopaminergic drugs in thestriatum possibly via pallidostriatal feedback loops. Neurosci.Lett. 240(3):167-170, 1998.

[0623] Mobilia, G., Zuin, G., Zanco, P., DiPede, F., Pinato, G., Neri,G., Cargnel, S., Ravile, A., Ferlin, G., Buchberger, R., 1998. Effectsof spinal cord stimulation on regional myocardial blood flow in patientswith refractory angina. A positron emission tomography study. G. Ital.Cardiol. 28, 1113-1119.

[0624] Molander, C., Q. Xu, C. Rivero-Melian, and G. Grant.Cytoarchitectonic organization of the spinal cord in the rat: II. Thecervical and upper thoracic cord. J.Comp Neurol. 289 (3):375-385, 1989.

[0625] Molenaar, I., A. Rustioni, and H. G. Kuypers. The location ofcells of origin of the fibers in the ventral and the lateral funiculusof the cat's lumbo-sacral cord. Brain Res. 78 (2):239-254, 1974.

[0626] Molenaar, I. and H. G. Kuypers. Cells of origin of propriospinalfibers and of fibers ascending to supraspinal levels. A HRP study in catand rhesus monkey. Brain Res. 152 (3):429-450, 1978.

[0627] Moravec, M., A. Courtalon, and J. Moravec. Intrinsicneurosecretory neurons of the rat heart atrioventricular junction:possibility of local neuromuscular feed back loops. J.Mol.Cell.Cardiol.18: 357-367, 1986.

[0628] Moravec, M. and J. Moravec. Intrinsic innervation of theatrioventricular junction of the rat heart. Am.J.Anat. 171: 307-319,1984.

[0629] Moravec, M. and J. Moravec. Adrenergic neurons and shortproprioceptive feedback loops involved in the integration of cardiacfunction in the rat. Cell Tissue Res. 258: 381-385, 1989.

[0630] Moravec, M., Moravec, J., and Forsgren, W. G. Catecholaminergicand peptidergic nerve components of intramural ganglia in the rat heart.Cell and Tissue Research 262, 315-327. 1990.

[0631] Murphy, D. A., O'Blenes, S., Nassar, B. A., and Armour, J. A.Effects of acutely raising intrathoracic pressure on cardiac sympatheticefferent neuron function. Cardiovascular Research 30, 716-724. 1995.

[0632] Murphy, D. A., G. W. Thompson, J. L. Ardell, R. McCraty, R. S.Stevenson, V. E. Sangalang, R. Cardinal, M. Wilkinson, S. Craig, F. M.Smith, J. G. Kingma, and J. A. Armour. The heart reinnervates aftertransplantation. Ann.Thorac.Surg. 69 (6):1769-1781, 2000.

[0633] Nadeau, R., D. Lamontagne, R. Cardinal, J. de Champlain, and J.A. Armour. Coronary sinus norepinephrine concentrations duringventricular tachycardia induced by left stellate ganglion stimulation indogs. Can.J.Physiol Pharmacol. 66 (4):419-421, 1988.

[0634] Norrsell, H., Eliasson, T., Albertsson, P., Augustinsson, L. -E.,Emanuelsson, H., Eriksson, P., Mannheimer, C., 1998. Effects of spinalcord stimulation on coronary blood flow velocity. Coron. Artery Dis. 9,273-278.

[0635] Nozdrachev, A. D. and A. G. Pogorelov. Extracellular recording ofneuronal activity of the cat heart ganglia. J.Auton.Nerv.Syst. 6: 73-81,1982.

[0636] Oldfield, B. J., and E. M. McLachlan. Localization of sensoryneurons traversing the stellate ganglion of the cat. J.Comp Neurol. 182(4 Pt 2):915-922, 1978.

[0637] Oppenheimer, S. M. and D. A. Hopkins. Suprabulbar neuronalregulation of the heart. In Armour, J. A. and J. L. Ardell, eds.Neurocardiology. New York, Oxford University Press. 1994, 309-342.

[0638] Page P. L., N. Dandan, P. Savard, R. Nadeau, J. A. Armour, and R.Cardinal. Regional distribution of atrial electrical changes induced bystimulation of extracardiac and intracardiac neural elements.J.Thorac.Cardiovasc Surg. 109 (2):377-388, 1995.

[0639] Paintal, A. S. A study of right and left atrial receptors. J.Physiol. 120: 596-610, 1953.

[0640] Parati, G., J. P. Saul, M. Dr Rienzo, and G. Mancia. Spectralanalysis of blood pressure and heart rate variability in evaluatingcardiovascular regulation: a critical appraisal. Hypertension 25:1276-1286, 1995.

[0641] Paulus, W. J., Grossman, W., Serizawa, T., Bourdillon, P. D.,Pasipoularides, A., Mirsky, I., 1985. Different effects of two types ofischemia {haeck over (Z)} on myocardial systolic and diastolic function.Am. J. hysiol. Heart. Circ. Physiol. 248, H719-H728.

[0642] Pauza, D. H., Skripka, V., Pauziene, N., and Stropus, R.Anatomical study of the neural ganglionated plexus in the canine rightatrium: Implications for selective denervation and electrophysiology inthe sinoatrial node in dog. The Anatomical Record 255, 271-294. 1999.

[0643] Pelleg, A. Cardiac cellular electrophysiologic actions ofadenosine and adenosine trisphosphate. Am. Heart J. 110: 688-693, 1985.

[0644] Perkel, D. H., G. L. Gerstein, and G. P. Moore. Neuronal spiketrains and stochastic point processes. I. The single spike train.Biophys.J. 7 (4):391-418, 1967.

[0645] Perkel, D. H., G. L. Gerstein, and G. P. Moore. Neuronal spiketrains and stochastic point processes. II. Simultaneous spike trains.Biophys.J. 7 (4):419-440, 1967.

[0646] Pinto, J. M. and P. A. Boyden. Electrical remodeling in ischemiaand infarction. Cardiov.Res. 42: 284-297, 1999.

[0647] Plecha, D. M., Randall, W. C., Geis, G. S., and Wurster, R. D.Localization of vagal preganglionic somata controlling sinoatrial andatrioventricular nodes. American Journal Physiology 255, R703-R708.1988.

[0648] Poree, L. R., and L. P. Schramm. Role of cervical neurons inpropriospinal inhibition of thoracic dorsal horn neurons. Brain Res. 599(2):302-308, 1992.

[0649] Potter, E. Presynaptic inhibition of cardiac vagal postganglionicnerves by neuropeptide Y. Neurosci.Lett. 83: 101-106, 1987.

[0650] Priola, D. V. and H. A. Spurgeon. Cholinergic sensitivity of thedenervated canine heart. Circ.Res. 41: 600-606, 1977.

[0651] Qin, C., M. J. Chandler, K. E. Miller, and R. D. Foreman.Chemical activation of cervical cell bodies: effects on responses tocolorectal distension in lumbosacral spinal cord of rats.J.Neurophysiol. 82 (6):3423-3433, 1999.

[0652] Qin, C., M. J. Chandler, K. E. Miller, and R. D. Foreman.Responses and afferent pathways of superficial and deeper c(1)-c(2)spinal cells to intrapericardial algogenic chemicals in rats.J.Neurophysiol. 85 (4):1522-1532, 2001.

[0653] Randall, D. C., Brown, D. R., Li, S. G., Olmstead, M. E.,Kilgore, J. M., Sprinkle, A. G., Randall, W. C., and Ardell, J. L.Ablation of posterior atrial ganglionated plexus potentiates sympathetictachycardia to behavioral stress. American Journal Physiology 275,R779-R787. 1998.

[0654] Randall, W. C., Armour, J. A., Geis, G. S., and Lippincott, D. B.Regional cardiac distribution of sympathetic nerves. FederationProceedings 31, 1199-1208. 1972.

[0655] Randall, W. C., J. L. Ardell, D. Calderwood, M. Milosavljevic,and S. C. Goyal. Parasympathetic ganglia innervating the canineatrioventricular nodal region. J.Auton.Nerv.Syst. 16: 311-323, 1986.

[0656] Randall, W. C., J. L. Ardell, R. D. Wurster, and M.Milosavljevic. Vagal postganglionic innervation of the canine sinoatrialnode. J.Auton.Nerv.Syst. 20: 13-23, 1987.

[0657] Randall, W. C. and J. L. Ardell. Functional anatomy of thecardiac efferent innervation. In Kulbertus, H. E. and G. Franck, eds.Neurocardiology. Mount Kisco, N.Y., Futura Publishing Co., Inc. 1988,3-24.

[0658] Randall, W. C. and J. L. Ardell. Nervous Control of the Heart:Anatomy and Pathophysiology. In Zipes, D. P. and J. Jalife, eds. CardiacElectrophysiology: From Cell to Bedside. Philadelphia, W. B. SaundersCompany. 1990, 291-299.

[0659] Randall, W. C. Efferent sympathetic innervation of the heart. InArmour, J. A. and J. L. Ardell, eds. Neurocardiology. New York, N.Y.,Oxford University Press. 1994, 77-94.

[0660] Randich, A., and W. Maixner. Interactions between cardiovascularand pain regulatory systems. Neurosci.Biobehav.Rev. 8 (3):343-367, 1984.

[0661] Randich, A., and S. A. Aicher. Medullary substrates mediatingantinociception produced by electrical stimulation of the vagus. BrainRes. 445 (1):68-76, 1988.

[0662] Randich, A., and G. F. Gebhart. Vagal afferent modulation ofnociception. Brain Res.Brain Res.Rev. 17 (2):77-99, 1992.

[0663] Randich, A. Neural substrates of pain and analgesia. ArthritisCare Res. 6 (4):171-177, 1993.

[0664] Ranson, S. W. Afferent paths for visceral reflexes. Physiol. Rev.1: 477-522, 1921.

[0665] Ren, K., A. Randich, and G. F. Gebhart. Vagal afferent modulationof a nociceptive reflex in rats: involvement of spinal opioid andmonoamine receptors. Brain Res. 446 (2):285-294, 1988.

[0666] Ren, K., A. Randich, and G. F. Gebhart. Vagal afferent modulationof spinal nociceptive transmission in the rat. J.Neurophysiol. 62(2):401-415, 1989.

[0667] Ren, K., A. Randich, and G. F. Gebhart. Electrical stimulation ofcervical vagal afferents. I. Central relays for modulation of spinalnociceptive transmission. J.Neurophysiol. 64 (4):1098-1114, 1990.

[0668] Ren, K., A. Randich, and G. F. Gebhart. Effects of electricalstimulation of vagal afferents on spinothalamic tract cells in the rat.Pain 44 (3):311-319, 1991.

[0669] Riley, D. A. Effects of neuropeptides on heart rate in dogs:comparison of VIP, PHI, NPY, CGRP, and NT. American Journal Physiology255, H311-H317. 1988.

[0670] Robb, J. S. Comparative Basic Cardiology. Grune & Stratton, NewYork, 1965.

[0671] Roudenok, V., L. Gutjar, V. Antipova, and Y. Rogov. Expression ofvasoactive intestinal polypeptide and calcitonin gene-related peptide inhuman stellate ganglia after acute myocardial infarction. Ann.Anat. 183:341-344, 2001.

[0672] Rouleau, J. R., Simard, D., Kingma Jr., J. G., 1999. Myocardialblood flow regulation relative to left ventricle pressure and volume inanesthetized dogs. Can. J. Physiol. Pharmacol. 77, 902-908.

[0673] Rowe, Brian P., Saylor, David L., and Speth, Robert C. Analysisof Angiotensin II Receptor Subtypes in Individual Rat Brain Nuclei.Neuroendocrinology 55, 563-573. 1992.

[0674] Rowell, R. B. Human Cardiovascular Control. New York, OxfordUniversity Press. 1993.

[0675] Saito, Kazuto, Potter, William Z., and Saavedra, Juan M.Quantitative autoradiography of b-adrenoceptors in the cardiac vagusganglia of the rat. European Journal of Pharmacology 153, 289-293. 1988.

[0676] Sanderson, J. E., Brooksby, P., Waterhouse, D., Palmer, R. B.,and Neubauer, K. Epidural spinal electrical stimulation for severeangina: a study of its effects on symptoms, exercise tolerance anddegree of ischemia. European Heart J. 13, 628-633. 1992.

[0677] Sanderson, J. E., Ibrahim, B., Waterhouse, D., and Palmer, R. B.Spinal cord stimulation for intractable angina: long term clinicaloutcome and safety. European Heart Journal 15, 810-814. 1994.

[0678] Sanderson, J. E., Woo, K. S., Chung, H. K., Chan, W. W., Tse, L.K., White, H., 1996. The effect of transcutaneous electrical nervestimulation on coronary and systemic hemodynamics in syndrome X. Coron.Artery Dis. 7, 547-552.

[0679] Sandkuhler, J., B. Steizer, and Q. G. Fu. Characteristics ofpropriospinal modulation of nociceptive lumbar spinal dorsal hornneurons in the cat. Neuroscience 54 (4):957-967, 1993.

[0680] Sandkuhler, J., and A. A. Eblen-Zajjur. Identification andcharacterization of rhythmic nociceptive and non-nociceptive spinaldorsal horn neurons in the rat. Neuroscience 61 (4):991-1006, 1994.

[0681] Sandkuhler, J., and A. A. Eblen-Zajjur. Identification andcharacterization of rhythmic nociceptive and non-nociceptive spinaldorsal horn neurons in the rat. Neuroscience 61 (4):991-1006, 1994.

[0682] Sandkuhler, J., A. Eblen-Zajjur, Q. G. Fu, and C. Forster.Differential effects of spinalization on discharge patterns anddischarge rates of simultaneously recorded nociceptive and non-nociceptive spinal dorsal horn neurons. Pain 60 (1):55-65, 1995.

[0683] Sandkuhler, J.. Neurobiology of spinal nociception: new concepts.Prog Brain Res. 110:207-224, 1996.

[0684] Sasayama, S., Nonogi, H., Miyazaki, S., Sakurai, T., Kawai, C.,Eiho, S., Kuwahara, M., 1985. Changes in diastolic properties of theregional myocardium during pacing-induced ischemia in human subjects. J.Am. Coll. Cardiol. 5, 599-606.

[0685] Savard, P., R. Cardinal, R. A. Nadeau, and J. A. Armour.Epicardial distribution of ST segment and T wave changes produced bystimulation of intrathoracic ganglia or cardiopulmonary nerves in dogs.J.Auton.Nerv.Syst. 34 (1):47-57, 1991.

[0686] Schaper, W. The Pathophysiology of Myocardial Perfusion.Amserdan, New York, Oxford, Elsevier/North-Holland Biomedical Press.1979.

[0687] Schmidt, H. H. H. W., Schurr, C., Hedler, L., and Majewski, M.Local modulation of noradrenaline release in vivo: presynapticβ₂-adrenoceptors and endogenous adrenaline. J.Cardiovascul.Pharmacol.6(4), 641-649. 1984.

[0688] Schoebel, F. C., Frazier, O. H., Jessurun, G. A. J., DeJongste,M. J. L., Kadipasaoglu, K. A., Heintzen, M. P., Jax, T. W., Cooley, D.A., Strauer, B. E., and Leschke, M. Refractory angina pectoris inend-stage coronary artery disease: evolving therapeutic concepts.American Heart Journal 134, 587-602. 1997.

[0689] Schramm, L. P., and R. H. Livingstone. Inhibition of renal nervesympathetic activity by spinal stimulation in rat. Am.J.Physiol 252 (3Pt 2):R514-R525, 1987.

[0690] Schwartz, P. J., M. Pagani, F. Lombardi, A. Malliani, and A. M.Brown. A cardiocardiac sympathovagal reflex in the cat. Circ.Res. 32(2):215-220, 1973.

[0691] Schwartz, P. J., R. D. Foreman, H. L. Stone, and A. M. Brown.Effect of dorsal root section on the arrhythmias associated withcoronary occlusion. Am.J.Physiol 231 (3):923-928, 1976.

[0692] Schwartz, P. J., G. E. Billman, and H. L. Stone. Autonomicmechanisms in ventricular fibrillation induced by myocardial ischemiaduring exercise in dogs with a healed myocardial infarction: anexperimental preparation for sudden cardiac death. Circulation 69:780-790, 1984.

[0693] Schwartz, P. J., E. Vanoli, M. Stramba-Badiale, G. M. De Ferrari,G. E. Billman, and R. D. Foreman. Autonomic mechanisms and sudden death.New insights from analysis of baroreceptor reflexes in conscious dogswith and without myocardial infarction. Circulation 78: 969-979, 1988.

[0694] Schwartz, P. J., M. T. La Rovere, and E. Vanoli. Autonomicnervous system and sudden cardiac death. Experimental basis and clinicalobservations for post-myocardial infarction risk stratification.Circulation 85: I77-I91, 1992.

[0695] Schwartz, P. J., and M. T. La Rovere. ATRAMI: a mark in the questfor the prognostic value of autonomic markers. Autonomic Tone andReflexes After Myocardial Infarction. Eur.Heart J. 19 (11):1593-1595,1998.

[0696] Seabrook, G. R., L. A. Fieber, and D. J. Adams. Neurotransmissionin neonatal rat cardiac ganglion in situ. Am.J.Physiol. 259: H997-H1005,1990.

[0697] Selyanko, A. A. Membrane properties and firing characteristics ofrat cardiac neurons in vitro. J.Auton.Nerv.Syst. 39: 181-190, 1982.

[0698] Selyanko, A. A. and V. I. Skok. Synaptic transmission in ratcardiac neurones. J.Auton.Nerv.Syst. 39: 191-200, 1992.

[0699] Sgambato, V., N. Maurice, M. J. Besson, A. M. Thierry, and J. M.Deniau. Effect of a functional impairment of corticostriataltransmission on cortically evoked expression of c-Fos and zif 268 in therat basal ganglia. Neuroscience 93 (4):1313-1321, 1999.

[0700] Shvalev, V. N. and Sosunov, A. A. A light and electronmicroscopic study of cardiac ganglia in mammals. Z Mikrosk Anat Forsch99, 676-694. 1985.

[0701] Skok V. Physiology of Autonomic Ganglia. Tokyo: I. Shoin, Ltd.,1973.

[0702] Smith, F. M., D. A. Hopkins, and J. A. Armour.Electrophysiological properties of in vitro intrinsic cardiac neurons inthe pig (Sus scrofa). Brain Res.Bull. 28: 715-725, 1992.

[0703] Smith, F. M., A. S. McGuirt, D. B. Hoover, J. A. Armour, and J.L. Ardell. Chronic decentralization of the heart differentially remodelscanine intrinsic cardiac neuron muscarinic receptors. Am.J.Physiol.HeartCirc.Physiol. in press, 2001.

[0704] Smith, F. M., A. S. McGuirt, J. Leger, J. A. Armour, and J. L.Ardell. Effects of chronic cardiac decentralization on functionalproperites of canine intracardiac neurons in vitro.Am.J.Physiol.Regulatory Integrative Comp.Physiol. in press, 2001.

[0705] Smith M L, Thames M D. Cardiac receptors: dischargecharacteristics and reflex effects. In: Armour J A, Ardell J L, eds.Neurocardiology. New York: Oxford University Press, 1994:19-52.

[0706] Smith, M. V., A. V. Apkarian, and C. J. Hodge, Jr. Somatosensoryresponse properties of contralaterally projecting spinothalamic andnonspinothalamic neurons in the second cervical segment of the cat.J.Neurophysiol. 66 (1):83-102, 1991.

[0707] Steedman, W. M., and S. Zachary. Characteristics of backgroundand evoked discharges of multireceptive neurons in lumbar spinal cord ofcat. J.Neurophysiol. 63 (1):1-15, 1990.

[0708] Sternini, C. and N. Brecha. Distribution and colocalization ofneuropeptide Y- and tyrosine hydroxylase-like immunoreactivity in theguinea-pig heart. Cell Tissue Res. 241: 93-102, 1985.

[0709] Stiller, C. O., Cui, J. -G., O'Conner, W. T., Brodin, E.,Meyerson, B. A., Linderoth, B., 1996. Release of GABA in the dorsal hornand suppression of tactile allodynia by spinal cord stimulation inmononeuropathic rats. Neurosurgery 39, 367- 375.

[0710] Summers, Roger J., McMartin, Lynne R., Kompa, Andrew R., Gu,Xinhua, and Molenaar, Peter. Signalling pathways in cardiac failure.Clinical and Experimental Pharmacology and Physiology 22, 874-876. 1995.

[0711] Sylven, C. Angina pectoris. Clinical characteristics,neurophysiological and molecular mechanisms. Pain 36, 145-167. 1989.

[0712] Tanaka, S., K. W. Barron, M. J. Chandler, B. Linderoth, and R. D.Foreman. Low intensity spinal cord stimulation may induce cutaneousvasodilation via CGRP release. Brain Res. 896 (1-2):183-187, 2001.

[0713] Tejani-Butt, S. M. [³H]Nisoxetine: A radioligand for quantitationof norepinephrine uptake sites by autoradiography or by homogenatebinding. Journal Pharmacology and Experimental Therapeutics 260,427-436. 1992.

[0714] Thies, R. and R. D. Foreman. Descending inhibition of spinalneurons in the cardiopulmonary region by electrical stimulation of vagalafferent nerves. Brain Res. 207 (1):178-183, 1981.

[0715] Thompson, G. W., Hoover, D. B., Ardell, J. L., and Armour, J. A.Canine intrinsic cardiac neurons involved in cardiac regulation possessNK1, NK2 and NK3 receptors. American Journal Physiology 275,R1683-R1689. 1998.

[0716] Thompson, G. W., K. Collier, J. L. Ardell, G. Kember, and J. A.Armour. Functional interdependence of neurons in a single canineintrinsic cardiac ganglionated plexus. J.Physiol 528: 561-571, 2000.

[0717] Thompson, G. W., M. Horackova and J. A. Armour. Chemotransductionproperties of nodose ganglion cardiac afferent neurons in guinea-pigs.Am. J. Physiol., 279: R433-R439, 2000.

[0718] Thoren, P. Characteristics of left ventricular receptors withnonmedullated vagal afferents in cats. Circulation Research 40, 415-421.1977.

[0719] Thorén, P. Role of cardiac vagal c-fibers in cardiovascularcontrol. Rev. Physiol. Biochem. Pharmacol. 86: 1-94, 1979.

[0720] Uchida, Y., and S. Murao. Afferent sympathetic nerve fibersoriginating in left atrial wall. Am.J.Physiol 227 (4):753-758, 1974.

[0721] Uchida, Y., and S. Murao. Excitation of afferent cardiacsympathetic nerve fibers during coronary occlusion. Am.J.Physiol 226(5):1094-1099, 1974.

[0722] Urban, L. and R. E. Papka. Origin of small primary afferentsubstance P-immunoreactive nerve fibers in the guinea-pig heart.J.Auton.Nerv.Syst. 12: 321-331, 1985.

[0723] Vance, W. H., and R. C. Bowker. Spinal origins of cardiacafferents from the region of the left anterior descending artery. BrainRes. 258: 96-100, 1983.

[0724] Van der Velde, E. T., Burkhoff, D., Steendijk, P., Karsdon, J.,Sagawa, K., Baan, J., 1991. Nonlinearity and load sensitivity ofend-systolic pressure-volume relation of canine left ventricle in vivo.Circulation 83, 315-327.

[0725] Vanoli, E., G. M. DeFerrari, M. Stramba-Badiale, S. S. Hull, R.D. Foreman, and P. J. Schwartz. Vagal stimulation and prevention ofsudden death in conscious dogs with a healed myocardial infarction.Circ.Res. 68: 429-435, 1991.

[0726] Vanoli, E. and P. B. Adamson. Baroreflex sensitivity: methods,mechanisms and prognostic value. PACE 17: 434-445, 1994.

[0727] Vanoli, E., S. S. Hull, Jr., P. B. Adamson, R. D. Foreman, and P.J. Schwartz. K+ channel blockade in the prevention of ventricularfibrillation in dogs with acute ischemia and enhanced sympatheticactivity. J.Cardiovasc Pharmacol. 26 (6): 847-854, 1995.

[0728] Vanoli, E., P. B. Adamson, Lin Ba, G. D. Pinna, R. Lazzara, andW. C. Orr. Heart rate variability during specific sleep stages. Acomparison of healthy subjects with patients after myocardialinfarction. Circulation 91 (7):1918-1922, 1995.

[0729] Vegh, A., Szekeres, L., Parratt, J. R., 1991. Transient ischemiainduced by rapid cardiac pacing results in myocardial preconditioning.Cardiovasc. Res. 25, 1051-1053.

[0730] Verburgh, C. A., J. Voogd, H. G. Kuypers, and H. P. Stevens.Propriospinal neurons with ascending collaterals to the dorsal medulla,the thalamus and the tectum: a retrograde fluorescent double-labelingstudy of the cervical cord of the rat. Exp.Brain Res. 80 (3):577-590,1990.

[0731] Warner, M. R. and M. N. Levy. Neuropeptide Y as a putativemodulator of the vagal effects on heart rate. Circ.Res. 64: 882-889,1989.

[0732] Warner, M. R., P. D. Senanayake, C. M. Ferrario, and M. N. Levy.Sympathetic stimulation-evoked overflow of norepinephrine andneuropeptide Y from the heart. Circ.Res. 69: 455-465, 1991.

[0733] Watson-Wright, W., G. Boudreau, R. Cardinal, and J. A. Armour.Beta 1- and beta 2-adrenoceptor subtypes in canine intrathoracicefferent sympathetic nervous system regulating the heart. Am.J.Physiol261 (5 Pt 2):R1269-R1275, 1991.

[0734] Watson-Wright, W. M., M. Wilkinson, D. E. Johnstone, R. Cardinal,and J. A. Armour. Prolonged supramaximal stimulation of canine efferentsympathetic neurons induces desensitization of inotropic responseswithout a change in myocardial beta-adrenergic receptors. Can.J.Cardiol.8 (2):177-186, 1992.

[0735] Watson-Wright, W. M., M. Wilkinson, R. Cardinal, G. Boudreau, andJ. A. Armour. Minimal modification of canine ventricular myocyte cellsurface beta adrenoceptors despite desensitisation of ventricularfunction during exogenous beta adrenoceptor challenge. Cardiovasc Res.28 (5):680-683, 1994.

[0736] Weber, R. N., R. W. Blair, and R. D. Foreman. Effects of cardiacadministration of bradykinin on thoracic spinal neurons in the cat.Exp.Neurol. 78 (3):703-715, 1982.

[0737] Wei, F., R. Dubner, and K. Ren. Nucleus reticularisgigantocellularis and nucleus raphe magnus in the brain stem exertopposite effects on behavioral hyperalgesia and spinal Fos proteinexpression after peripheral inflammation. Pain 80 (1-2):127-141, 1999.

[0738] Weihe, E., M. Reinecke, and W. G. Forssman. Distribution ofvasoactive intestinal polypeptide-like immunoreactivity in the mammalianheart: Interrelation with neurotensin- and substance P-likeimmunoreactive nerves. Cell Tissue Res. 236: 527-540, 1984.

[0739] Weinreich, D., G. M. Koschorke, B. J. Undem and G. E. Taylor.Prevention of the excitatory actions of bradykinin by inhibition of PGI₂formation in nodose neurones of the guinea-pig. J. Physiol. 483:735-746,1995.

[0740] Wharton, J. and S. Gulbenkian. Peptides in the mammaliancardiovascular system. Experientia 43: 821-832, 1987.

[0741] Wharton, J., J. M. Polak, L. Gordon, N. R. Banner, D. R.Springall, M. Rose, A. Khagani, J. Wallwork, and M. H. Yacoub.Immunohistochemical demonstration of human cardiac innervation beforeand after transplantation. Circ.Res. 66: 900-912, 1990.

[0742] White, J. C. Cardiac pain: Anatomic pathways and physiologicmechanisms. Circ. 16: 644-655, 1954.

[0743] Wijffels, M., C. Kirchhof, R. Dorland, J. Power, and M. A.Allessie. Electrical remodeling due to atrial fibrillation inchronically instrumented conscious goats. Roles of neurohumoral changes,ischemia, atrial stretch and high rate of electrical activation.Circulation 96: 3710-3720, 1997.

[0744] Wilkinson, M., A. Giles, J. A. Armour, and R. Cardinal.Ventricular, but not atrial, M2-muscarinic receptors increase in thecanine pacing-overdrive model of heart failure. Can.J.Cardiol. 12(1):71-76, 1996.

[0745] Wilson, Stephen K. Peripheral Alpha-1 and Alpha-2 AdrenergicReceptors in Three Models of Hypertension in Rats: An In VitroAutoradiography Study. Journal Pharmacology and ExperimentalTherapeutics 256, 801-810. 1991.

[0746] Xi-Moy, S. X., W. C. Randall, and R. D. Wurster. Nicotinic andmuscarinic synaptic transmission in canine intracardiac ganglion cellsinnervating the sinoatrial node. J.Auton.Nerv.Syst. 42: 201-214, 1993.

[0747] Xi, X., W. C. Randall, and R. D. Wurster. Morphology ofintracellularly labeled canine intracardiac ganglion cells.J.Comp.Neurol. 314: 396-402, 1991.

[0748] Xi, X., W. C. Randall, and R. D. Wurster. Intracellular recordingof spontaneous activity of canine intracardiac ganglion cells.Neurosci.Lett. 128: 129-132, 1993.

[0749] Xi, X., J. X. Thomas, W. C. Randall, and R. D. Wurster.Intracellular recordings from canine intracardiac ganglion cells.J.Auton.Nerv.Syst. 32: 177-182, 1991.

[0750] Xu, Z. J. and D. J. Adams. adrenergic modulation of ioniccurrents in cultured parasympathetic neurons from rat intracardiacganglia. J. Neurophysiol. 69: 1060-1070, 1993.

[0751] Yakhnitsa, V., Linderoth, B., Meyerson, B. A., 1999. Spinal cordstimulation attenuates dorsal horn neuronal hyperexcitability in a ratmodel of mononeuropathy. Pain 79, 223-233.

[0752] Ye, F., S. Zangenehpour, and A. Chaudhuri. Light-induceddown-regulation of the rat class 1 dynein-associated proteinrobl/LC7-like gene in visual cortex. J.Biol.Chem. 275 (35):27172-27176,2000.

[0753] Yuan, B. X., J. L. Ardell, D. A. Hopkins, and J. A. Armour.Differential cardiac responses induced by nicotine sensitive canineatrial and ventricular neurones. Cardiovasc.Res. 27: 760-769, 1993.

[0754] Yuan, B. X., J. L. Ardell, D. A. Hopkins, and J. A. Armour.Differential cardiac responses induced by nicotine sensitive canineatrial and ventricular neurons. Cardiov.Res. 27: 760-769, 1993.

[0755] Yuan, B. X., Ardell, J. L., Hopkins, D. A., Losier, A. M., andArmour, J. A. Gross and microscopic anatomy of the canine intrinsiccardiac nervous system. The Anatomical Record 239, 75-87. 1994.

[0756] Zhang, J., M. J. Chandler, and R. D. Foreman. Thoracic visceralinputs use upper cervical segments to inhibit lumbar spinal neurons inrats. Brain Res. 709 (2):337-342, 1996.

[0757] Zhang, J., M. J. Chandler, K. E. Miller, and R. D. Foreman.Cardiopulmonary sympathetic afferent input does not require dorsalcolumn pathways to excite C1-C3 spinal cells in rats. Brain Res. 771(1):25-30, 1997.

[0758] Zimmermann, M. Encoding in dorsal horn interneurons receivingnoxious and non noxious afferents. J.Physiol (Paris) 73 (3):221-232,1977.

[0759] Zoubina, E. V. and P. G. Smith. Sympathetic hyperinnervation ofthe uterus in the estrogen receptor alpha knock-out mouse. Neurosci.103: 237-244, 2001.

[0760] Zucker, I. H., W. Wang, M. Brandle, and H. D. Schultz. Baroreflexand cardiac reflex control of the circulation in pacing-induced heartfailure. In Spinale, F. G., ed. Pathophysiology of Tachycardia-InducedHeart Failure. Armonk, N.Y., Futura Publishing Company. 1996, 193-226.

[0761] Zucker I H. Reflex Control of the Circulation in Heart Failure.In: Shepherd J T, Vatner S F, eds. Nervous Control of the Heart.Amsterdam: Harwood Academic Publishers, 1996:357-378.

1. A method for protecting cardiac function and reducing the impact ofischemia on the heart, comprising the steps of: providing a stimulatorcapable of generating a predetermined electrical signal; placing thestimulator adjacent a neural structure capable of carrying thepredetermined electrical signal from the neural structure to theintrinsic cardiac nervous system; and activating the stimulator for apredetermined period of time to generate the predetermined electricalsignal to protect cardiac function and reduce the impact of ischemia onthe heart.
 2. The method of claim 1, wherein the neural structure is aspinal cord.
 3. A method for treating an animal having a cardiacpathology by protecting cardiac function and reducing the impact ofischemia on the heart, comprising the steps of: providing a stimulatorcapable of generating a predetermined electrical signal; placing thestimulator adjacent a neural structure capable of carrying thepredetermined electrical signal from the neural structure to at leastone of the intrinsic cardiac nervous system and the heart; andactivating the stimulator for a predetermined period of time to generatethe predetermined electrical signal to modulate at least one of theintrinsic cardiac nervous system and the heart, and thereby protectingat least one of the intrinsic cardiac nervous system and the heart totreat the cardiac pathology.
 4. The method of claim 3, wherein theneural structure is a spinal cord.
 5. A method for electricallycommunicating with at least one of an intrinsic cardiac nervous systemand a heart, comprising the steps of: providing a stimulator capable ofgenerating a predetermined electrical signal; placing the stimulatoradjacent a neural structure capable of carrying the predeterminedelectrical signal from the neural structure to at least one of theintrinsic cardiac nervous system and the heart; and activating thestimulator for a predetermined period of time to generate thepredetermined electrical signal to communicate with at least one of theintrinsic cardiac nervous system and the heart.
 6. The method of claim5, wherein the neural structure is a spinal cord.
 7. A method ofmodulating electrical neuronal and humoral responses of at least one ofan intrinsic cardiac nervous system and a heart, comprising the stepsof: providing a stimulator capable of generating a predeterminedelectrical signal; placing the stimulator adjacent a neural structurecapable of carrying the predetermined electrical signal from the neuralstructure to at least one of the intrinsic cardiac nervous system andthe heart; and activating the stimulator for a predetermined period oftime to thereby generate the predetermined electrical signal to modulatethe electrical neuronal and humoral response of at least of theintrinsic cardiac nervous system and the heart.
 8. The method of claim7, wherein the neural structure is a spinal cord.
 9. A method ofactivating spinal cord neurons to induce a conformational change in anintrinsic cardiac nervous system, comprising the steps of: providing astimulator capable of generating a predetermined electrical signal;placing the stimulator adjacent a spinal cord to carry the predeterminedelectrical signal from the spinal cord to an intrinsic cardiac nervoussystem; and activating the stimulator for a predetermined period of timeto thereby generate the predetermined electrical signal to therebyactivate spinal cord neurons in proximity of the stimulator so as toinduce a conformational change in the intrinsic cardiac nervous system.10. The method of claim 9, wherein the neural structure is a spinalcord.
 11. A method for the prolonged activation of spinal cord neuronsto induce a conformational change in an intrinsic cardiac nervoussystem, comprising the steps of: providing a stimulator capable ofgenerating a predetermined electrical signal; placing the stimulatoradjacent a spinal cord to carry the predetermined electrical signal fromthe spinal cord to an intrinsic cardiac nervous system; and activatingthe stimulator for a predetermined period of time to thereby generatethe predetermined electrical signal to thereby activate spinal cordneurons in proximity of the stimulator so as to induce a conformationalchange in the intrinsic cardiac nervous system wherein the activationeffects persist for a period of time extending beyond the activation ofthe stimulator.
 12. The method of claim 11, wherein the neural structureis a spinal cord.
 13. A method for transiently nullifying neuronalactivation of an intrinsic cardiac nervous system by myocardialischemia, comprising the steps of: providing a stimulator capable ofgenerating a predetermined electrical signal; placing the stimulatoradjacent a neural structure capable of carrying the predeterminedelectrical signal from the neural structure to the intrinsic cardiacnervous system; and activating the stimulator for a predetermined periodof time to thereby generate the predetermined electrical signal totransiently nullify neuronal activation of an intrinsic cardiac nervoussystem by myocardial ischemia.
 14. The method of claim 13, wherein theneural structure is a spinal cord.
 15. A method for prolongednullification of neuronal activation of an intrinsic cardiac nervoussystem by myocardial ischemia, comprising the steps of: providing astimulator capable of generating a predetermined electrical signal;placing the stimulator adjacent a neural structure capable of carryingthe predetermined electrical signal from the neural structure to theintrinsic cardiac nervous system; and activating the stimulator for apredetermined period of time to thereby generate the predeterminedelectrical signal to nullify the neuronal activation of the intrinsiccardiac nervous system by myocardial ischemia for a prolonged period oftime extending beyond stimulator activation.
 16. The method of claim 15,wherein the neural structure is a spinal cord.
 17. A method fortransiently suppressing neuronal activation of an intrinsic cardiacnervous system by myocardial ischemia, comprising the steps of:providing a stimulator capable of generating a predetermined electricalsignal; placing the stimulator adjacent a neural structure capable ofcarrying the predetermined electrical signal from the neural structureto the intrinsic cardiac nervous system; and activating the stimulatorfor a predetermined period of time to thereby generate the predeterminedelectrical signal to transiently suppress the neuronal activation of theintrinsic cardiac nervous system by myocardial ischemia.
 18. The methodof claim 17, wherein the neural structure is a spinal cord.
 19. A methodfor prolonged suppression of neuronal activation of an intrinsic cardiacnervous system by myocardial ischemia, comprising the steps of:providing a stimulator capable of generating a predetermined electricalsignal; placing the stimulator adjacent a neural structure capable ofcarrying the predetermined electrical signal from the neural structureto the intrinsic cardiac nervous system; and activating the stimulatorfor a predetermined period of time to thereby generate the predeterminedelectrical signal to suppress the neuronal activation of the intrinsiccardiac nervous system by myocardial ischemia for a prolonged period oftime extending beyond stimulator activation.
 20. The method of claim 19,wherein the neural structure is a spinal cord.